Membrane Display of a Fusion Protein containing the icenudeation

protein from Pseudomonas synngae and ScFv against c-myc

oncoprotein in recombinant Eschecichia coli

Daniel N- Ding

Department of Chernical and Biochemical Engineering

Faculty of Engineering Science

Submitted in partial fulfillment

of the requirernents for the degree of

Master of Engineering Science

Faculty of Graduate Studies

The University of Western Ontario

London, Ontario

Apd, 1999

@Daniel N. Ding 1999 .

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Abstract

The ice nucleation protein (INP) is a glycosyl phosphatidylinositol

anchored outer membrane protein found in certain Gram negative bacteria. In

this study, the INP from Pseudomonas syringae was applied as a fusion partner

with the single chain antibody fragment (Scfv) against the human onco-protein c-

myc. The objectives were to hvestigate whether this approach could be utilized

for the surface display on E- coli of the a c-myc scFv.

An E. coli INP surface expression vector pNinaZ was first constructed

which contained the multicloning sites hcluding BsrGI at its centra1 repeating

region and BsaAl at its unique C terminal region for subcloning of foreign genes.

The gene fragment encoding the ScFv a c-mye was obtained from a c-myc

hybridoma ceIl line using the Recombinant Antibody Phage System (RAPS) from

Pharmacia. This was in-frame inserted into the BsrGl and BsaAl sites of pNinaZ

plasmid.

The two new constructed plasmids were named pNinaZ-myc and

pNinaZScFv-BsaAl- Both plasmids were cloned into E. coli host cell JM109. The

expression of fusion ScFv-INP protein was successfully demonstrated by SDS-

PAGE. The expression of the fusion proteins had no effect on cell viability as

revealed b y growth studies under various conditions. Measurement of ice

nucleation activity revealed that the INP portion of the fusion ptotein was

presented on the outer surface of the E. coli outer membrane as expected. Flow

cytometry studies of E. coli cells expressing the fusion proteins were conducted

following immunostaining or fluorescent binding of antigen peptides. Such

studies indicated that the ScFv portion of the fusion protein was predominantly

on the periplasmic face of the outer membrane. lncreasing the porosity of the

outer membrane by EDTA treatrnent increased the acœssibility of the ScFv

protein to the fiuorescent antigen. Alternative sites for insertion of the ScFv

iii

ScFv fragment into the IN? protein are considered for future studies to hopefully

direct the ScFv fragment to the outer face of the outer membrane. This study has

demonstrated for the first time. the application of INP as a transport motif for

ScFv a c-myc.

Key words: Ice nucleation: Fusion proteins; ScFv; c-myc

To rny beloved wife Ah Ji

Your are the wind beneath rny wings

Acknowledgements

I would like to express rny snicere gratitude to my chief supem-sor.

Dr. Amaqeet Bassi and CO-supervisors Dr. Greg Gloor and Dr. Argyriyrios

Margaritis. for giving me the opportunity to accomplish this research and for al1 of

their guidance in doing it-

1 would like to thank Michelle Gaasenbeek of the Molecular Biology

Laboratory. Department of Biochernistry. UWO for her technical assistance in the

early stage of this research. Many thanks go to Dr- Greg Gloor for allowïng me to

do this research in that laboratory. Also thanks to Tarnmy Ofay. Angela Coveny

and Faye Males of that labcratory for their help and friendship through the

realization of this research.

I wouid ais0 like to thank Or. Steven Lindow of UC Berkeley for providing

plasmid pUC18131CE; Dr. Stanley Dunn of Department of Biochernistiy, UWO for

providing plasrnrd pSD: Mr. Martin White of John Robarts Institute for his

technical assistance in fiow cytometry analysis. Ms- Anne Brickenden of

University Hospital. London for her technical advice in my immunoassay.

This research was supported by an NSERC Individual Research Grant to

Dr. A. Bassi. Partial support for the project was also provided by Dr. A. Margaritis'

research grant. A special UWO scholanhip from the Faculty of Engineeeng

Science. UWO to me is also gratefully acknowledged.

Table of Contents

.. ...................................................................... Certificate of Examination.. II ... ............................................................................................. Abstract ..III

Acknowledgernents ......................................................................................... vi

Table of Contents ............................................................................................... vii

........................................................................................................ List of Tables x

List of Figures ...................................................................................................... xi ... ..................................................................................................... Nornencfature XIII

Chapter 1 Introduction ...................................................................................... 1

............................................................................................... 1 -1 Introduction 1

................................................................................................. 1 -2 Objectives 3 ............................................................................ C hapter 2 Literature Review 4

............................................... 2.1 Principies of recombinant DNA technoiogy 4

2.1.1 Definitions: DNA . RNA . gene and protein ........................................... 4

2.1 -2 DNA cloning and cloning vectors ........................................................ 5

....................................... 2.1 -3 Fusion p rotein expression by E . coii vecton 6

2.2 Principles of bacterial ice nucleation ......................................................... 8

2.2.1 Introduction ................. .. ...................................................................... 8

2.2.2 Physicai basis of ice nucleation .......................................................... 8

2.2.3 The ice nucleation genotype and phenotype .................................... 10

........... 2.2.4 Ice nucleation protein bcalization and their structure models 13

......................................................... 2.3 Prïnciples of antibody engineering 1 5

2.3.1 Antibodies ......................................................................................... 15

............................................................................ 2.3.2 Antibody structure 16

........................................................................... 2.3.3 Antibody fragments 18

.................................. 2.3.4 Scfv (single-chain Fragment variable) design 19

............................... 2.4 Fusion protein expression on the surface of E . coii 21

.................... 2-43 Different fusion protein surface expression motifs ........21

vii

2.4.2 Choice of transporting motif for the expression of ScFv fusion

......................................................... protein on the surface of coli 24

...................................................................... 2.5 Flow cytometry and FACS 25

...................... Chapter 3 Experimental MaterÏals and Methods .... .................. -27

3.1 Bacteriai strains . plasmids and culture conditions ................................... 27

3.2 Construction of surface display vectors .............................................. 31

....... 3.2.1 Construction of the INP E- coli surface display vector - pNha2 31

.................... 3.2.2. Preparation of DNA fragment eocoding ScF v a<-myc 34

3.2.3 Construction of intermediate prasmids ......................................... -... 35

3.2.4 Construction of the INP-c-myc fusion protein coli surface

................................................................ display vector pNinaZ-rnyc 38

3.2.5 Canstruction of the INP-c-myc fusion protein coii surface

display vector - pNinaZScFv-BsaAl .................................................. 40

3.3 GrowthstudiesofE.coiihostcellXL-1. JM109 .................................... 42

........................................ 3.3.1 Effect of growth medium and temperature 42

............................... 3.3.2 Growth studies of E- coli host cell XL-1 . JM 109 43

.................................................. 3 -4 Measurement of ice-nucieation actjvity -44

.............................................. 3.5 SDS-PAGE and Western blotting anaiysis 45

.......... ...................................................*.................... 3.5.1 SOS-PAGE ,,... 45

3.5.2 Western blotting ................................................................................ 45

3.6 Flow cytometry ....................................................................................... 47

......................................................... 3.6.1 1 mmunostaining of the bacteria 47

3.6.2 Preparation of fluorescent antigen .................................................... 49

Chapter 4 Results and Discussion ..................................................................... 50

4.1 introduction ....... ....... .. ........................................................................... 50

4.2 Construction of surface display vecton .................................................. 50

....... . 4.2.1 Construction of the INP E coii surface display vecior - pNinaZ 50

4.2.2. Preparation of DNA fragment encoding ScFv a-c-mye .................... 53

4 - 2 3 Construction of intermediate plasmids .................................... 2 4.2.4 Construction of the I NP-c-myc fusion protein E- coii surface

display vector pNinaZ-rnyc ......... ................................................ 55 .

viii

4.2.5 Construction of the NP-c-rnyc fusion protein coii surface

dispfay vector - pNinaLScFv-BsaAL ....................................... ,. ........ 57

4.3 Growth studies of E . colt celf . XL-1 . JM 109 .......................................... 59

........................................ 4.3.1 Effect of growth medium and temperature 59

4.3-2 GrowUi studies of host cells JM i 09 containhg pNinaZ . pNinaZ-myc and pNinaZScFv-BsaAl ................................................ 66

4.4 Measurement of ice-nucfeation activity ................................................... 68

4.5 SOS-PAGE and Western blotting anafysis .............................................. 70

4.5.1 SDS-PAGE ...................................................................................... 7 0

4.5.2 Western blotting ............................................................................... 71

4.6 Flow cytornetry ........................................................................................ 72

......................................................... 4.6.1 Imrnunostaining of the bacteria 72

Chapter 5 Conclusions and Recommendations ................................................. 77

References- ....................................................................................... -79 Appendix A Experimental Protocols ................................................................. 88

Appendix A 1 GEN ECLEAN" (Short Protocol)(B IO 1 0 1 Inc. . Vista . CA) ............ 88

Appendix A2 Transformation of E . co/i (D . Hanahan) ................................... 89

Appendix A3 Cracking method .............................................................. -90

Appendix A4 Rapid boiling method for plasmid DNA (Hofmes and Quigley) ...... 91

Appendix A5 Western blotting ............................................................... -92

Appendix A6 irnmunodetection .......................................................... ..... 94

VITA .................................................................................................. 96

List of Tables

Table 2.1 Maximum Size of DNA That Can Be Cloned in Vectors ............----- 6

List of Figures

Figure 2.1 a) A simpie E . coii expression vector utilizing the lac prornoter

b) An E . coli expression vector utilizing the lac prornoter for INP

........................................................................... protein expression 7

Figure 2.2 Typical INP primary structure ...................................................... 1 2

Figure 2.3 Typical structure of an antibody molecule ......................................... 17

Figure 2.4 The antibody fragments ................................................................... 18

................................ Figure 2.5 The fluorescence-activated celf sorter (FACS) -26

Figure 3.1 The plasmid map of pSD .................................................................. 28

Figure 3.2 The plasmid map of psluescript II SK (+/-) ....................................... 29

Figure 3.3 The plasmid map of pK18h ............................................................... 30

Figure 3.4 The plasmid rnap of pNinaZ .............................................................. 33

Figure 3.5 The plasmîd rnap of pNinaZ-myc ............................................... .......39

Figure 3.6 The plasmid map of pNinaZScFv-BsaAl ........................................... 41

Figure 4.1 Gel Quantitation of Purified ScFv Fragment ...................................... 53

Figure 4.2 Cell growth of XL-1 and JM 109 in SOB (@ 37O C ........................... 59

Figure 4.3 Viable cell numbers of XL-1 and JM 109 in SO6 @ 37" C .............. 60

Figure 4.4 Cell growth of JM109 in SOB @37 OC in rotating shaker (rpm 15) .. 61

Figure 4.5 Cell growth cornparison of JM109 @ 37°C in SOB and LB ............... 62

Figure 4.6 Viable cefl numbers of JM 109 in SOB and LB @ 37' C .................. 63

Figure 4.7 Ceil growth cornparison of JM109 in LB @ 22°C and 37°C .............. 64

Figure 4.8 SDS result of INP and INP u ScFv c-myc expression @37'C

........................................................................................... and 22°C 65

Figure 4.9 Cornparison among JM109 containing pSD . pNinaL pNlna2-WC

and pNinaZScFv-BsaAl in SOB @ 37°C ........................................... 66

Figure 4.1 O Viable cell numbers of JM1O9 containing pSD . pNinaZ . pNlnaZ-myc and pNinaZScFv-BsaAl in SOB @ 37OC ...................... 67

Figure 4.11 a) INP activities of JM109 in LB @WOc counted after 2 minutes

... b) INP actïvities of JM109 in LB @37Oc counted after 4 minutes -69

Figure 4.12 6% SDS-PAGE .......................................-....-.......-........................... 70

Figure 4.1 3 Flow cytometfical analysis of E-coîi expressing pSD. pNïnaL

............ pNinaZ-rnyc that are stained by FITC-anti Mouse IgG (H+L) 73

Figure 4.14 Flow cytometrkaf analysis of E-coii expressing pNinaZ.

pNinaZ-myc stained by fluorescent c-rnyc peptide and treated by

EDTA @O°C on ice ..............-......................--.................................... 74

Figure 4.15 Flow cytornetricaf analysis of Ekoiïexpressing pNinaZ.

pNinaZ-myc and pNinaZScFv-BsaAl stained by FM-c-myc peptide

and treated by EDTA @Tc.. .........-......-.............................................. 75

xii

Nomenclature

63 Ab1

Ab2

ATCC

bp

BSA

CH

CL

C-term inal

DD

DMF

DNA

cDNA

dNTP

EDTA

Fab

Fc

FACS

Fv

FITC

FM

Gfy

IgG

I na

/na'

!NP

I PTG

kD

first Iinked antibody

second linked antibody

American Type Culture Collection

base pair(s)

bovine serurn aibumin

constant-region heavy chain

constant-region light chain

carboxyt-terminal

double distilled

N,N-dirnethyformamide

deoxyribonucleic acid

corn plemen ta ry DNA

3'deoxyribonucleotide-5'-triphosphate

ethylenediarninetetraacetic acid

fragment antigen binding

fragment crystalline

fluorescence assisted cell sorting

fragment variable

fluorescein isothiocyanate

ffuoresceinmaleimide

glycine

immunoglobulin G

Ice nucleation activity

Ice nucleation activity positive

ice nucleation protein

isopropylthiogaiactoside

kilo Dalton. (1 -66 x 10"' g)

xiii

MW

NIH

N-terminal

OD

PAGE

PBS

PCR

RNA

mRNA

rRNA

tRNA

ScFv

ScFv a

SDS

Ser

TE

Tr

Tris

VH

VL

rnofecular weight

National Institute of Health

amino-terminal

optical density

polyacrylamide gel electrophoresis

phosp hate-buffered saline

polyrnerase chain reaction

ribonucleic acid

messenger RNA

ribosomal RNA

transfer RNA

single chah antibody fragment variable

singie chain antibody fragment var~able against

sodium dodecyl sulfate. CH~(CH~)I iSO4 N a

Serine

Tris-EDTA buffer solution

room temperature

Tris (hydroxymethyl) aminomethane

varia ble-reg ion heavy chain

variable-region light chain

xiv

Chapter 1 lntroduction and Objectives

1 -1 Introduction

Many aerobic Gram-negative strains of bacterial species, such as

Pseudumonas syringae. Pseudomonas fluorescens and EmMa henbkoIa.

possess an outer membrane protein which can ~atalyze ice formation h m supercoded water (Lindw. 1983). The protein which is responsiMe fœ iœ

nucleation is named ice nucfeation protein (NP). The INP resides on the sufaœ

of cells (Wobler. 1993) and is stable in the stationary phase of the culture

(Nemecek-Marshall et al-. 1993). Genes conferring ice nucleation activity have

been sequenced from five bacteriai species and al1 encode proteins with similar

structure (Abe et al., 1989; Green and Warren. 1985; Warren and Corotto, 1989;

Warren et al., 1986; Zhao and Orser. 1990). The central repetitive region of the

INP encoding gene inaZ. from Pseudomonas syffngae. has a good toi- for

DNA rearrangements while retaining the ice nucleation activity if such

rearrangements do not change the reading frame (Green et aL, 1988). Those

unique characteristics make the INP an ideai fusion protein to allow the

expression of useful enzymes and antibody fragments in the bacterial outer

membrane.

It has been demonstrated that the expression levels of 62 kD human

nuclear oncoprotein myc (c-rnyc) (Evan et al.. 1985) are elevated in transfomed

cells (Evan et al.. 1986). The ovarexpression of cnyc was shown to strongly

correl ate with rew rrence and survival rates of breast carcinoma (Papamichalis et

a 1988; Bland et al., 1995). A similar prognostic value was suggested for

human hepatoceflular carcinoma (Tiniakos et a/-, 1989). This general finding has

bwn proven of diagnostic value in various types of cancer, making us8 of the higher expression of c-myc causeci by the higher proliferative rate.

The monoclonal antibody a c-myc recogniang the cmyc oncopmtein has

been shown to be useful in a broad range of analyses dealing with the det-on

of the c-myc protein (Gloor et al-, 1995). Cunently, an ELISA technique is used

for detedion of c-myc which is based on the a C-myc antibody- Hwver , this

antibody is very expensive. A 200 gg a ccnyc antibody kit costs US$ 289.00

(Upstate Biotechnofogy lnc-. Lake Plaüd. New York. 1998).

The expression of proteins on the bactensl surface is important f a a

variety of biotechnological applications such as the development of live bactefial

vaccine. the display and screening of peptide and antibody librarïes, for bactefia-

based solid-phase imrnunoassays~ and finally for the production of wholecell

adsorbents (Georgiou et ai., 1993; Hofnung 1991 ). Another advantage of

expressing proteins on bacterial surface is that such proteins are no subject to

degradation by cytoplasmic proteases. Expression of proteins in a f m that is

exposed on the cell surface has also proven useful of studying the binding

specificity of periplasmic binding proteins. These applications would be faalitaed

by the availability of transportation motifs for expressing proteins to the bactefial surface.

A nurnber of €.colt fusion protein surface display have been accomplished

with a variety of surface display motifs including Lam6 (Charbit et al.. 1988);

PhoE (Agterberg et al.. 1990); OmpA (Francisco et al., 1992); PAL (Fuchr et al..

1991 ; 1996 and Taylor et ai.. 7 990); ice-nucieation protein (INP)(Jung et al.,

1998a; 1998b) and an N-teminally attached passenger protein expmssion

system (Maurer et al.. 1997; Handerson et al., 1998). Two of them. PAL (Fuchs

et al.. 1 991 ) and OmpA (Franchco et al., 1993) have been desaibed for the

bacterial display of antibody fragments. Hawever. besides the fad that anübody-

PAL had little effect on E.mk cell growth and viability, both systerns had size limitations for foreign DNA fragment insertion which Iirnited their applications fot

other fusion pmtein expression (Chen and Heming , 1 987; Georgiou et el., 1 998).

1.2 Objectives

The following were the goals and objectives of this study:

a) Construct an cok INP surface expression vector using the KMZ gene

from Pseudomonas synngae-

b) Constmct the ScFv a c-myc gene fragment from hybridoma cell Iines

expressing c-myc-

C) C ~ n ~ t n t t t a plasmid containing the fusion protein expression s y d m

comprising of inaZ gene and ScFv a c-myc gene fragment.

d) Transfomi this expression system in E. co/i host ceils and optîrnize the

incubation conditions.

e) lnvestigate the expression of fusion proteins from cloned plasmids with

different insertion site and optimize the expression ability.

f) Evaluate the efficiency of the expression for its possibility to be used in

further biosensor or bioseparation applications.

Chapter 2 Literature Review

2.1 Principfes of recombinant ONA technology

2.1 -1 Definitions: DNA RNA. gene and protein

DNA (deoxyribonucleic aQd) is the hefeditary molecule in ail ceYular life

foms, as weil as in many vinises. It is a long linear polymer, composa9 of four

kinds of deoxyribose nudeotides Iinked by phosphodiester bonds. In its native

state. DNA is a double helix composed of two antiparalfel pofynucleotide stfards.

RNA (ribwiudeic acid) is composed of ribose nucleotides linked by

phosphodiester bonds. RNA is f o n d by transcription of DNA or, in soma

viruses. by the copying of RNA There are three types of cellular RNA - mRNA,

rRNA. and tRNA and they play different roles in protein synthesis.

A gene is the entire DNA sequence necessary for the synthesis of a

functional polypeptide or RNA molecule. It is the unit of inheritance, which cames

information from one generation to the next.

A protein is the linear polymer of amino acids Iinked together by peptide

bonds in a specific sequence. Proteins play a very important rote in the biology

of cells. They served as enzymes, structurai elernents, antibodies. hormones etc.

They exhibit four levels of structure: prirnary (the sequence of amino aüds),

secondafy. tertiary, and quatemary.

The reiationship between DNA, RNA and protein is:

2.1 -2 DNA cloning and cloning vecton

DNA cloning is the recombinant DNA technique in which specific cDNAs

or fragments of genomic DNA are ïnserted into a cloning vedor. an

autonomously replicating DNA moiecule. which then is incorporated into cultured

host ceil (e-g.. E. coli ceils) and maintained during growth of the host cella This

enables the DNA segment to be replicated with Me vedor.

Several cloning vecton indude plasmid, bactetiophage k, wsmid, P l

vector and YAC (yeast artficial chromosame) are used. Table 1 s h a m the

maximum size of DNA that can be cloned in vectors.

Among above vectors, E-cali pfasmids are the most cornmonly used ones

in recombinant ONA technology. However, these plasrnids are diffefent from their

naturaliy occorring ones. They have been engineered to optimize their use as

vectors in DNA cloning. Their length is nomaily reduced to only about 3 kb,

which is much less than that of naturally occumng E. coli plasrnids.

The most cornmonly used Ecoli expression vectors are namally

assembled by the ligation of two parts: 1) A basic plasmid vector which

containing a replication ohgin, a dntg-resistance gene and a foteign DNA donhg

region. 2) A promoter, which is a region of DNA involved in binding of RNA

polymerase to initiate transcription.

Table 2-1 Maximum Size of DNA That Can Be Cloned in Vectocs

(Adopted from Lodish et al.. 1 995)

( Vector Type Length of Cloned DNA (kb) 1 Plasmid 20

, Bacteriophage A 25 I

Cosmid 45

P l vector 100 I

YAC (yeast artificial chromosome) 1000

2- 1 -3 Fusion protein expression by E- coli vectors

If the inserted foreign ON A has a protein-cading sequence and its reading frame is kept the same as the plasmid protein, a fusion protein will be

synthesized. That fusion protein wiil have its N-teminal encoded by the plasmid

and the rernainder encoded by the inserted DNA-

A kind of typical E. coli plasmid used for fusion protein expression

contains a cioned E- coii chromosome that includes the fac promoter and the

cDNA gene encoding the intended protein. Lactose. or a lactose analog such as

isopropylthiogalactoside (IPTG), is needed for the mRNA transcription fmm the

lac promoter. IPTG is more widely used cause it cannot be metabolized. Its

concentration does not change as the cells grow. After addition of IPTG. the fe gene is transcfibed into rnRNA, which then is translated to yield many copies of

the intended protein. Figure 2.1 a) shows such a plasmid with lac2 gene encodng

P-galadosidase.

If a cDNA enwding INP replaces the lac2 gene, a new plasmid for the

production of ice nudeation protein (INP) is constnicted. Figure 2.1 b shows the

lacZ gene is cut out of the plasmid with restriction enzyme and replaœd by the

[NP cDNA. When the resulting plasmid is transfomied into E. colil addition of

IPTG and subsequent transcription from the promoter produces INP mRNA.

which is translateci into INP. In this proœss, the lac promoter, which is required

for efficient transcription, must be maintained just before the start site of the

inserted cDNA.

lac

- IPTG t IPTG

l a d g e n e

Figure 2.1 a) A simple E. coli expression vector utilizing the lac promoter

b) An E. coli expression vector utilizing the lac promoter for INP

protein expression

(Adopted and modifieci from Lodish et al., 1995)

2.2 Principies of bacterial ice nucleation

Some bacteria of the genera Pseudomonas, Eiwinia and Xanthomnas

possess proteins that enable them to nucleate the ~rystallization of ice from

supercooled water (Dye et al., 1380; Hirano et al., 1978; Paulin and Luisetti

1978; Lindow et ai.. 1978a: Lindow et al.. 1978b; Yankofsky et al., 1 mis). Pseudomonas sytfngae is one of the species which has been widely studicid. The

annually frost damage caused by Pseudomonas syffngae to plants and associated agricultural crops is estimated about billion dollars wa(dmde.

However new beneficial applications of ice-plus bacteria also have been

continually developed. e.g., the manufacture of snow and the freeze textufhg of different foods (Margaritis and Bassi, 1991) and reœntly the fusion profeins

comprising the INP and the enzyme levansucrase and carboxyrnethylœ~lulase

respectively have been reported (Jung et ai.. 1998a: 1998b)-

2-22 Physical basis of ice nudeation

2.2.2-1 Types of ice nudeation

If ice and pure water are combined at O°C and without energy change, the

two phases wilf exist in equBibrium. Water will ainvert to ice only if heat is

removed. No nucleation is needed for this phase change. Aff water m3fItually

becomes ice while a temperature of Oo couid be maintained. However. if there is

initielly no ice in the system (water only), the solidification follows a very dillbrent

path. Upon cooling, the temperature of this system eventually draps bekw the

freering point (supercooling) before nucleation and furtrier solidification can proceed, as nucleation must precede crystal gmwth. This sugge- that

nucleation is a necessary step to overcome an energy (activation energy) before

the conversion.

Three mechanisms of ice nucleation are comrnonfy recognized (Fletcher, 1970). They are:

homogeneous ice nucieation (self-nucieation of ice uystallîzation in pure

supercool ed water)

heterogeneous ice nudeation (initiation of ice crystallization through binding

of supercooled water of some non-water material)

secondary ice nucfeation (seeding of ice crystallization by a pre-existing ice

crystal)

2.2.2.2 Measurement of icenucfeation activity

Ice-nucleation activity is usually rneasured via Vali's method (Vali. 1971 )

which is very similar to the rnost probable number method (Finney, 1984) used in

rnicrobioiogy. Nucleatîon actïvity is calwlated from the frequency of freezïng

observed in multiple. small volumes (often droplets. Say 40 x 10 VI) of a sample

which are diluted in water or buffer sofution at the measurement tempersture,

where some but not al1 of Vie droplets get frozen. Generally, freezîng is cwnted

visually, either by monitoring the color of the droplets changed from transparency

to translucence which accompanies freezing, or by inclusion of a patented

fluorescent freezing indicator dye (Warren and Wolber, 1 988).

It is obsenred that bacterial ice nuclei are active at widely variable

frequencies and temperaturas. and that the variation is rnodulated by bacterial stfain and growth conditions.

Three classes of bacterial ice nudei were used to define this variation (Yankofsky et a/.. 1 981 b)- They are:

type I nuciei. which are active at temperatures between -2OC and -5%

type II nuclei, which are active at temperatures batween -5OC and -7OC

type 111 nudei, which are adive at -7% or lower temperatures

Just as the most probable number method in rnicrobiology assumes no

background (Le. sterile media), measurement of ice nucleation assumes that the

diluent and test surface are free frorn ice nuciei active at or above the assay temperature. In practice. bMer solution wuld be Type I nuclei free through

autoclave or microfiltration, and nudeus-free surfaces can be easily achievlied by

spray-coating alurninurn foil boat with a 2% solution of parafin in xylene, and

then baking the foi1 at f OPC, The afuminum foi1 boats are placed in a

temperature controfled environment, for example, foating on the surface of a

reftigerated bath containing chilied refrigerant (ethanol or a 50:50 ethylene glycol

and water mixture).

2.2.3 The ice nucleation genotype and phenotype

The bactenai ice nucleation study was greatly launched by the discovery

that the ha' phenotype could be transferred ta Escherichia coli by cloning a

single stretch of dromosomaf DNA from an /na' bacterial species (Orner et al.,

1985). To date, ai least ten DNA fragments capable of imposing the /na+ phenotype to Ecoli have been cloned and the DNA sequences of five of the

fragments have been published (Orser et ai.. 1985; Amy et al.. 1976; Green and

Warren, 1 985; Corotto et al., 1 986; Yankofsky et ai-, 1983; Arai et ai.. 1989; Zhao

and Orser, 1990; Anderson and Ashworth, 1986; Hasegawa et ai.. 1990; Schrnid

et al., 1 997).

The five sequenced DNA fragments shared several properties:

ail contain one long open-reading frame of -3600 bp.

about 80% of every open-reading frame consists of a series of

hierarchically organized, irnperfectly repeated DNA sequences with

fangVis of 24.48 and 144 bp.

the DNA sequences of the ope~eading frames are highly homologous

between each other, whiie the DNA sequence of the regions outside the

open-reading frames are usually not homologous-

protein sequences inferred front DNA sequences are more ~Vongly

conserved than the DNA sequenœs thernselves (Le. rnany of the base

changes among genes are silent differences at redundant positions in the

codons of the open-reading frames)-

Studies of transposon-insertion mutation of ice-nucfeation clones (Orser et

al., 1985; Corotto et al., 1986). sequencing of the N-terminus of the pmtein

product of another sudi clone (Wolber et al.. 1986) and studies of the effeds of

heterologous promoters on expression of the Ina' phenotype (Wolber et al-.

1986; Southworth et al.. 1 988) have provided additional evidence that, in every

micro-organism, the [na' phenotype is the result of expression of a single ice-

nucleation gene, to yield a single ice-nucleation protein.

fhe five sequenced /na' genes encode five proteins with highly

homologous inferred amino acid-residue sequences. The sequences can be

organized into a series of domains. based upon the absence or preS8nœ of

particular types of repeated arnino acïd-residue sequences (Green and Warren, 1985; Warren et a/.. 1986. 1987; Warren and Corottu, 1989; Wolber and W J ~ ~ ,

1991 ). The consensus domain structure

nucleation genes is outlined (Figure 2.2).

inferred from the five sequenced ice-

- - --- - c m - -- --

a-- -- - - - --- ---

40 1 6 8 1 48 1 L8 1 4 8 - 1 4 8 1 4 8 1

Figure 2.2 Typical INP primary structure

(Adapted from Wobler and Warren, 1 989)

The majority of the amino acid sequenœ of each ice-nudeating protein

(81%) was found to be a cornplex repeating domain that could be further

subdivided into three regions. The first two regions repeat with a high fidelity 2

periodicity of 48 residues. Each of these 48-mer units in the first two repeating

ragions consisted of three 16-mer units with medium fidelity repeats. The 16mer

units were themselves composed of two repeating $-mer units, AGYGST - T and

A - - - S - - -, of low fidelity repeats. which means that only two posiüorts out of

eigM were strongly conserved. The third repeating section consisted only of 8-

mer repeating units. A - - - S - - -. as shown in Figure 2.2.

While the majority of the ice nucleation protein was a central repeang

region as described above. 1 5% of the sequence was unique N-terminal domain

and 4% fomed a c-terminal domain wique ta each protein from the three

different bacteria. The N-terminal domain was found to be relatively hydrophobie,

which would indicate membrane Iocafization whiie the C-terminal domain was

relativeiy hydrophilic.

Deletion mutation studies of the ha' gene. in which the remaining

sequences were in frame. was used to probe the function of the ice nudeation

protein. Removal of the N-teminal unique dornain caused the ice nudeaüon

activity of the P. synhgae bacteria to decrease with threshold temperatures t w r

than - 5 C Removal of the C-terminal unique dornain caused a total Io$$ of ice

nucleation adivity, while successive deletions in the repeating domain caused a

corresponding decrease in ice nudeation temperatures (Green et a', 1988).

2.2.4 Ice nucleation protein localization and their structure models

Lindow et al. (1989) has quantified the ice nucieation activity in different

sub-cellular fractions of P. symgae and Ecafi containhg the ice nudeation gene.

For both the rnicrocrganisrns, the im nudei were found to be localked in cell

envelopes. Ice nucleation advity was found in Triton4 1 00 insoluble membrane

fragments as well as in slowly precipitated high-density membrane fragments.

The outer membranes of these Gram-negative baderia had nearly all the ice

nucleation activity associated with them, No ice nucleation was detected in any

soluble cell fractions.

tf bacterial ice nuclei function as a result of a coherent ternplate

mechanism. then some part of the protein which foms such nuclei must f0ld to

fomi a surface which is capable of binding water in an amay t h a dosely

approximates a srnall ice crystal. The leading candidate for this tempfate is the

section of molecules containing repeated sequences.

The secondary structure of the central dornain is thought to be a B-pleated

sheet. purttnctuated by 5 to 6 turns per 48 amino acid sequence, the repeated unit

is hydrophific and particularfy ridi in serine and threonine- The most probable

tertiary structure of the central domain is a phairpin (Kajava and Lindow, 1993).

The later research results showed some support for this hypothetical mode1

(Schmid et ai., 1997). Rie secondary and teaiary structures of the Ngnd C-

terminal domains have not been modeled in any details- They were predicted to

consist of both a-helics and P-strands typical of globular proteins (Wanen et al.,

1986). However. the only portion of the molecule which contains hydrophobie

stretches which are candidates for conventional transmembrane a-helices is the

N-terminai unique domain- Thus, the N-terminal rnay contain the membrane

anchor of ice-nucleation proteins, and the ternplate formed by the repeating

domain may be associated only with the membrane surface. Such a ldon

would fit the postulated function of the repeating domain. namely organizing

water. Alternatively, ice-nudeation proteins could be anchored to lipid molecules

by some form of secondary chernical modification (Turner et al.. 1990, 1991,

Kozloff et a/., 1991). lt is still an unsolved problem as ta what the secondary and

tertiary structures of an ice-nudeatian protein are.

2.3 Principles of antibody engineering

2-3.1 Antibodies

Antibodies are fïrst defined functionafly in the 1890s as a semm substance

capable of conferring passive immunity to other animals. Foity years later it was

discovered that the y globulin fraction of senim protein contahed adbody

reactivity. Their primary function is to bind antigen through the antigm-ôÏnding

sites on their amino-terminal ends. They have many advantageous pfopefües

such as its high degree of selectivity and affinity for its ligand and the potentially

vast nurnber of different antibodies (more than 1 0 ~ ~ 1 1 " ) . The production.

engineering and cfinical application of antibodies were not well developed until

two important immunological breakthroughs were made. One was the

development of hybridorna technology in 1975, through which hykidoma

(imrnortalized antibody-secreting cells) could be generated against any antigen

(ligand and can secrete virtually limitless quantities of antigen-macüve

monoclonal antibodies. The other was the elucidation of the rnechanism of

antibody gene rearrangement. These immunological dixoveries, toge th^ with

recent advances in genetic engineering and biofogica f chemistry, have

empowered scientists to design new antibodies. It may even mate new

combining site shapes and chernical constitutions which are not yet discovered

by nature (e-g., metallo antibodies). Up to now, a number of antibodies has been

designed as rnofecular tools in researb. diagnosis and therapy.

2-3.2 Antibody structure

Antibody molecules are gfywproteins. mey are composed of four

polypeptide chains. two ideriticai "heavy" and two identical "light" (Figun, 2-3).

Each chain is composed of domanis; heavy chains contain four or five. aceording

to their class and light chains contain two. The light chain wnsists of abait 220

amino acids and has a molecular weight of about 25 kD. The heavy cWn is

made up of apptoximately 450-575 amina acïds (depending on the d a a of the

heavy chain) with a molecular weight of about 51-72 kD. Each a m contains one

complete light chain and the N-terminal end of the heavy chain. m i l e the base is

comprised of the C-terminai end of the heavy chain-

The heavy chain and light chains are composed of a series of building

blocks of globular domains that are each about 1 10 amino acids long. These

domains. called imrnunoglobulin domains, have a characteristic tertiary sûucture of two roughty parallel -pleated sheets that are joined by a disulfide (Sa) bond-

Each iight chain has two domains, Mile each heavy chain has either four a five

dornains. The Mt 110 amino acids of the N-terminal pomon of both the heavy

and light chains Vary considerably between antibodies of clifferhg specificity

hence designated the variable domains (Vn and VL, respectively). They contain

the antigen-binding sites or hypervariabie regions that are complementafy to. and

thus bind. the antigenic determinants. Whereas al1 rernainders of both chahs are

more conserved in amino aud saquence and are designated the constant region

(CH and CL), while each heavy chain has one variable domain and three or four constant domains. depending on its class WH + CH 1 + CH 2+ CH CH 411-

Figure 2.3 Typical structure of an antibody rnolecule

(Adapted from Ritter and Ladynman, 1995)

2-3.3 Antibody fragments

Selective proteolytic cleavage of irnrnunoglobulin molecules in the vicinity

of the interchain disulfide bond results in various types of fragments. Pepsin and

papain are two protease that cleave immunoglobulin molecules at diaraderistic

sites-

Fab Fc

Figure 2.4 The antibody fragments

(Adopted from Mayforth, 1 993)

As in Figure 2.4, The protease pepsin (dashed arrow) cleaves the

immunoglobulin molecule on the C-terminal side of the disulfide bonds that link

the two heavy chains together. generating an F(ab')= fragment and several

srnaller cleavage produas of the Fc portion. The F(ab')2 fragments is divalent

(Le.. contains two antigen-binding sites) and can cross-link antigen. If the

disulfide bond joining the two portions of the heavy chains together in the F(ab')2

fragment is cleaved, two Fab' fragments can be produced. Çurther deavage of

the Fab' fragment with pepsin can create an Fv (Ffagment variable) fragment

wmposed of a VH domain non-covalently bonded to the VL domain.

Enzyrnatrc cfeavage of an antibody with papain (solid a m ) on the N-

terminal side of the disuifide bond joining the immunoglobulin heavy chains

generates three fragments, two Fab (Fragment antigen binding) fragments and

one Fc (Fragment crystatline). The Fab fragment contains the complete Iight

chain. the MO N-terminal heavy-chain domains (VH + Cd). and a srnall part of

the hinge region. (Men separated from the Iight chain. this part of the heavy

chain is called the Fd fragment).

The Fv, Fab and fab' fragments have a single antigen-binding site. In

contrast, the F(ab')* fragments. like whole irnmunogiobulin moleades. are

divalent and hence can cross-link antigenic detenninants. 8y definition. Fab.

F(ab')2. and Fv irnmunoglobulins lack the Fc region. and. as a result of Ws. lack

effecior function. which rnay be an advantageous feature in certain situations.

For exampie. a variety of cell types (macrophages. basophils. mast cells. and

lymphocytes, etc.) express receptors specific for the Fc portion of some

irnmunoglobulins. Unlike the whole immunoglobulin molecules. the antigen-

binding antibody fragments wouid not "nonspecificaiiy" bind to these reœptas.

2.3.4 ScFv (singledain Fragment variable) design

A recent development in antibody tedinology is the production of single

chain antigen binding proteins. Recombinant antibody fragments offer

advantages over fragments derived through proteolytic cleavage. as extensive

purification is required to remove the produas derived fom the FC region- Ak0,

Fv fragments (comprised of just a VH and VL domain) are paf t i~~laf ly d i f k ~ l t t0

derive through proteolytic cleavage. Furthenore, since the vanable domains in a

Fv fragment are not covaientiy iinked together, sorne F v fragment tend to

disassociate when diluted (Pluckthun, 1990). Recombinant singlechah Fv

fragments have been geneticâlly engineered that consists of VL and VH domains

attached by Iinker peptides. They retain the same spe~ficity and general atfinity

as the parent monocfonal antibody.

The starting point is gettïng mANA from an antibody prodicing

hybridomas. established cell Iines or spleen-defived 8 lymphocytes. Since a

hybndorna expresses the heavy and Iight chain genes for a single antibody, it

therefore represents the most abundant and straightforward source from which

antibody genes can be cloned. Then cONAs are genefated from rnRM with

reverse transcription. The heavy and light chain antibody genes are ampliîied in

two separate pnmary PCR reactions. After the purification. the heavy and Iight

chain DNA products are assembled into a single gene using a DNA Iinker

fragment. The assembled antibody ScFv DNA fragment is amplified by second

PCR ampiification reaction.

Although the first single chain Fv proteins were desaibed 1 1 yean ago. in

1988 (Houston et ai., 1988: Bird et al.. 19û8). they engendered widespread

applications of single-chain immunotechnology to Fv regions from antibodies and

other members of the immunoglobulin superfamily (Huston et al., 1993).

2.4 Fusion protein expression on the surface of CM

2.4.1 Oifferent fusion protein surface expression motifs

A number of fusion protein expression motifs on the surface of E d i have

been accomplished. They indude a) Lam6 (Charôit et a/.. 1988); b) PhoE

(Agtemerg et al.. 1990); c) OmpA (Francism et al.. 1992; 1993); d) PAL (Fuchs

et al.. 1 991 ; 1 996 and Taylor et ai.. 1990); e) ice-nudeation protein (INP)(Jung et

ab, 1998a: 1998b) and f) an N-teninaliy attached passenger protein expression

system (Maurer et ai-, 1997; Henderson el a/., 1998). Their mechanisrns and

applications as weiI as iimitations are described as follows:

a) Lam6 is an outer membrane protein which can be used as a canier for the

expression of a wide variety of peptides in tems of length and sequence at the

surface of Eschenchia coli There is a permissive site on the gene emding

Lam6 which allowing foreign gene insertion. The resulting hybrid proteins

essentially keep their biological activities with inserts of up to about 60 amino

acid residues.

b) The phosphate-limitation-inducible outer-membrane protein (Ph0E) is a

major outer membrane protein of Escherichia coii The polypeptide spans the

membrane 16 times. thereby exposing 8 ragions at the ceil surface. Insertions in

these regions did not affect the biogenesis of the protein. The system is very

flexible, since insertions varying in length and nature can be made in different

œll-surface-exposed regions of PhoE protein. without intemng with the

assembly process into the outer membrane. However, sorne limitations were

encountered. e.g., insertion of eight or more hydrophobie arnino aad residues

affectad both the translocation process across the inner membrane and the

assembly process into the outer membrane. Also. the insertion of seW@nCBs

containing many charged residues resulted in accumuiation of precursor protein

in the cytopiasm.

c) OmpA tripartite fusion protein is composed of (i) the signal sequence and

first nine N-terminal amino acids of the mature major Eschenchia GQ/Ï IipOpotein.

(ii) amino auds 46459 of the outer membrane protein OmpA, and (iii) the

complete foreign protein sequenœ. It has been reporteci being used on bas-

lactamase (EC 3.5.2.6)(Francisco et al., 1992) and ScFv antibody (Francisco et

al.. 1993) surface expression. For ScFv fusion. it was expressed at a high level

and was shown to bind the hapten with high affinity and specifiaty. Whole ceil

EL1 SAS. fluorescence rnicroscop y, protease sensitivity, and flow cytometry al1

confirmed that the ScFv was anchored on the outer membrane and was

accessible on the surface. For Lppl-0mpA'-betalactamase fusion, protein had an

enzymatically active beta-lactamase and was found predominantly in Vie outer

membrane. lmmunofluoresœnce microscopy. the accessibility of the fusion

protein. the accessibility of the fusion protein to extemally added protease, and

the rates of hydrolysis of nitrocefin and peniciilin G by whole cells demonstrated

that a substantiaf fraction (20-30%) of the beta-lactamase domain of the fusion

protein was exposed on the extemal surface of E. co/i However th8 later

research showed that changes in the pemeability of the outer membrane due to

the protein over expression are an unavoidable consequenœ of displaying a

large penplasmic protein on the surface of E. coii (Georgiou et al.. 1 996).

d) Peptidoglycan associated Iipoprotein (PAL) is ais0 used to target

recombinant antibodies to the surface of Escherichia coii (Fuchs et a', 1991:

1996). fts peptidogfycan associated proteîn component, Mr 16600 (Chen and

Henning, 1987). is modified at the amino-teminal cysteine by a lipid moiety that

is integrated into the outer membrane. The fusion protein was able to bind

antigens and was tightly bound to the murein layef of the cell enveiope.

Imrnunofluoresœnce studies on unfixed cells shawed that functionaf antibodies

were accessible at the surface of intact bacteria. The antbody-PAL had littfe

effect on ceIl growth and viabiiity.

e) Ice-nucfeation protein (INP)

Ice-nucieation protein (INP), an outer membrane protein frorn

Pseudomonas syringae, is able to catalyze the ice crystal formation of

supercooled water. Using Pseudomonas syringae INP as an anchofing motif.

fundional display of the foreign proteins were investigated. Zymomonas mobifis

levansucrase (Levu) and carboxyrnethylceilulase (CMCase). on the swfac8 of

Eschenchia coii (Jung et al., 1998a; 199ûb). The gene fragment encoding Levu

lCMCase were inserted into a plasmid, able to overexpress INP in t h Ecoli

under the control of lac promoter. to replace the stop codon on the C-terminal of

INP. The cells expressing 1NP-LevUfINP-CMCase were found to retain both the

imudeat ion and whole-cell levansucrase enzyme/ ~arb~~ymethylC€?Ihla~e

activities. The surface localization was further verified by imrnunofluorssœnce

microscopy, fluorescence-activated cell sorting fIow cytometry and immumgold

electron microscopi~l examination. No growth inhibition or changes in th outer

membrane integrity were observad upon the induction of fusion protein synthesis.

Viability of the cells was also maintained over 48 hours in the stationary phase.

f The autotransporters, a farnily of secreted proteins fiom Gram-negative

bacteria, possess an overalf unifying structure cornprishg three fundindional

domains: the N-terminal leader sequence, the secreted mature pmtein

(passenger domain) and a C-terminal (beta-) domain that f o n s a beta-banei

pore to allow secretion of the passenger protein. A mode! of the beta-barrel

structure. proposed to be responsible for outer membrane translocation, served

as a basis for the construction of fusion pmteins containing hetmlogous

passengers. The N-terrninally attached passenger proteins were transloosted to

the surfaœ to Ecoli ompT mutant without a size limitation of proteins or gowth

defea of the cells (Maurer et al.. 1997). Surface exposure was ascewned by

enzyme-linked immunosorbent assay, imrnunofluorescen~ microscopy. and irnrnunogold elecfron miaoscopy using antisenun specific for the passmg8r

domains,

2.4.2 Choice of transporting motif for the expression of ScFv fusion protein on

the surfaœ of E. coli

In the above five fusion protein surface expression motifs, only PAL and

OmpA had been used for ScFv expression. But both of them have thdr own

disadvantage. Compared with other motifs, INP and a~totranSp0rterS it S88mS do

not have the problems of the small size limitation for foreign protein and gfowtll

defect of the cells-

[NP was therefore chosen as the transporting motif in this study for the

expression of ScFv fusion protein on the surface of E. coli.

2.5 Flow cytametry and FACS

A flow cytometer can idmtify different cells by messuring the light they

scatter. or the fluorescence they ernit, as they fiow through a laser beam; thus it

can sort out celk of e particular type from a mixture (Henenberg and Sweet,

1976). A fluorescencesdnrated cell sorter, or FACS (Figure 2.5). an instrument

based on fiow cytometry, can select one ceIl from thousands of other cells.

As shown in figure 2.5. the cuncentrated celf suspension would read with

a fluorescent antibody or dye, which in tum binds 10 a paflicle or moleatle such

as DNA. After that. the suspension is rnixed with a buffer, and the ceîls are

passed single-file through a laser beam. The fluorescent light emitted by each

cell is then measured simultaneously. From this the size and shape of the cell

can be detemined. The suspension is then passed through a nonle. which

forces the suspension to fom tiny droplets containing at most one single cd. At

the same tinte. each droplet is given an electnc charge with the amount

proportional to the measure of fluorescence of its cell. After that. dmplets with no

charge or different electric charges (due to different amounts of bound dye) are

separated by an electric field and collected. It takes only rnilliseconds to sort

each droplet. so up to 10 million cells can pass through the machine each hour.

In this way, cells having desired properties can be easily and efficiently

separated and then grown.

Nonfluorescent cet!

* Ftuarescent cell . ' draplets

j - - Nonfluorescent I

celi droptet

Nonflucrascent cell

Fluorescent cell

- .. - Sortecl chargea droplets " :, 2.' containmg a fi uarescent cet1

.* 8 * ** 0 , . ', * t,,; '

* c , , . u *

hop. w~lh <-* iesrer charge e$ ->b'mw - -: grester charge

Scattered lighr detector

-- 4

Figure 2.5 The fluorescence-activated cell sorter (FACS)

(Adopted from Lodish et al., 1995)

Chapter 3 Experimental Materials and Methods

3.1 Bacterial strains, pfasmids and culture conditions

Escherichia cofi XLI -Blue sup€44 hsdR17 r d 1 endAl gyrA46 thï relA1

Iac- F [proAB' lacP lacZAMlS Tnl O(tetr)] or JM1W recAl supA sbc815 hsdR4

rpsL thi ~(fac-proAB) F' LW36 pmAB* fa& f a c Z d M I q were useci as the

recombinant host.

The 3,727 bp DNA fragment was € M l digested from plasmid

pUC18131CE which was obtained from Dr. S. E. Lindow of UC Berkeley. A 3600

bp inaZ gene (GenBank accession number X03035) was contained in that

fragment. The pUC18131CE was made by ligating together the 837 bp XmnC

EmRl fragment of pUC18 and the 1890 bp EcoRI-Xmnl fragment of pUCI3

(Robert and McPherson, 1 987), generating pUC1813 first. then ligated with 3,727

bp DNA fragment which induded the inaZ gene in its EmRl digested site.

The gene source of antibody a<-myc was obtained from hybfidorna cell

rnyc-1-9E10.2 (ATCC CRL-1729) through The Recombinant Phage Antibody

System (RPAS) from Pharmacia Biotech (Piscataway. NJ).

Plasrnid vectors pSD (Figure 3.1). p6luescript II SK(+/-) (Figure 3.2) and

pK1 8h (Figure 3.3) were used in the surface display vedor constructions.

E.coli cells were grown in SOB medium, which contains Bacto-tryptone 20

g, yeast extract 5 g, NaCl 0.6 g and KCI 0.186 g/L or LB medium contains Batte-

tryptone 10 g, yeast extract 5 g, NaCl 10 glL. SOB and LB culture plate medium

were with addition of agar 15 g and Ampicillin 10 mg&. Ampicillin (1W pglml)

and Kanamycin (50 pghnl) were added respectively for selection of recombinant

cells. The temperature of the culture was maintained at 37OC unless otherwise

indicated. Cell growth was followed by rneasuring optical density at 600 nm

(Spectfonic 601, Milton Roy. PA) and viable cells were counted as colonies

plated on SOB or LE medium containing Ampicillin (100 pglml) after the

appropriate dilution.

Figure 3.1 The plasmid map of pSD

Figure 3.2 The plasmid map of pBluesaipt II SK (+/O)

(Adopted from Stratagene Catalogue, 1998)

Figure 3.3 The pfasmid map of pK18h

(Adopted from Pridmore, 1987)

3.2 Construction of surface disptay vectors

3.2.7 Construction of the INP E coli surface display vector - pNinaZ

The EwRl digested 3,727 bp DNA fragment of pUC18131CE was Iigaied

with NdellHindlll double digested pSD. The expriment procedures an,

described as foflows:

a) pUC18131CE was digestad by EcoRl and pSD was doubled digested by

NdellHind If (The digestion reactions always followed the instnidions by

the restriction enzyme rnanufacturers unless otherwise indicated).

b) The 3,727 bp ONA fragment was made blunt-ended with dNTPs and gene

cleaning by the GENECLEAN@KIT from B I 0 101 Inc.. Vista. CA (Appendix

A l ). The Ndellifindlll double digested pSD was made blunt-endeâ and

then dephosphoryisted by shrimp al kaline phosphatase.

Typical blunt-ended making procedures were:

DNA digestion solution: 10-20 pl

dNTPs: 2 pl @ 1.0 rnM each

Klenow Fragment: 0.5 pl

. incubate 30 min. @ T,

. inactivate 65 min. @65OC

Typicat dephosphorylation procedures were:

DNA solution from above digestion or bluntsnded making

shrimp alkaline phosphatase 2 - 3 pl

incubate 30 min. @37OC

c) After overnight ligation. the ONA was transformecl into Ecoli host ceIl XL-1

based on the O. Hanahan rnethod (Appendix A2) and plated on Ampicillin'

SOB culture plate.

Typical ligation procedures were:

Digested DNA A (intendad fragment): 4 -6 4

Oigested DNA 8 (intended plasmid): 2 - 4 @

T4 Iigase: 1 PI

T4 bufFer: 1 ~ 1

DO water O-2pl

incubate ovemight @ Tr

d) The overnight wltured colonies were p i&d for cracking (Appendix A3)

for screening of the recombinant plasrnids.

e) The picked colonies were inocuiated in SOB medium and incubated

ovemight.

fl The ovemight cultures were used for DNA extradion through rapid boiling

methad (Holmes and Quigley, Appendix A4).

g) The extracted DNAs were digested by BssHll for orientation identification.

h) The selected recombinant plasmids were sent for sequencing.

i) The DNA sequence was compared with the GenBank for confirmation.

J) The new INP Emli recombinant plasmid was narned as pNinaZ (Figure

3.4).

k) The predicted five unique cutting sites, 437 Hpai, 503 Sa, 1634 Aalll.

2657 Sacl and BsffiI, available on inserted inaZ gene were confirmeci by

their individual digestions.

1 - 1 3,600 bp inaZ

Figure 3.4 The plasmid map of pNinaZ

3.2.2. Preparation of DNA fragment encoding ScFv arcmyc

The DNA fragment encoding ScFv acniyc was prepared from rnyc

hybridoma cell by the Recombinant Phage Antibody System (RPAS) from

Phamacia Biotech (Piscataway, NJ). This system is designed to clone mouse

antibody genes and to express and detect fundional antibodies. It is designed in

a flexible madularfamat with three parts:

Mouse ScFv Module

Expression Module

Oetection Module

In ouf experiment only Mouse ScFv Module and part of the Expmssion

Module has been usad* The experiment procedures are described as ~ O ~ ~ O W S :

(for detaileâ experimental protocois, please refer to Ovewiew of Recombinant

Phage Antibody S ystem (XY438-00-08, rev.6, Phamacia Biotech. PiSCataway,

NJ), Instmdions of ~u i ck~ rep " mRNA Purification Kit (XY-039-00-O?, fev.4) and

instructions of Mouse ScFv ModulelRecombinant Phage Antibody System (XY-

025-00-1 O, rev-4).

a) mRNA Purification: mRNA was isolated from rnyc hybridoma cell through

QuickPrepQ3 mRNA Purification Kit-

b) First-Strand cONA Synthesis: First-strand cDNA was synthesùed fm mRNA primed with random hexameis by reverse transmptase.

C) Primary K R : The 340 bp heavy and 325 bp light chah an~body gme

fragments were amplified in two separate PCR reactions.

Gel Quantitation of Primary PCR Products: The amplified heavy and Iight

chain fragments are purifed separately by agame gel elecbophoresis-

Purification of the DNA from the agarose gel was done by MiaoSpinm

Colurnns provided with the kit

Assernbly of the Heavy and Light Chains: A (Gly4 S W ) ~ linker fragment

was used for amealing the $'-end of the heavy chah to the Send of the

light chah. The ScFv DNA fragment was -750 bp in length.

Second PCR Amplification Reaction: The ScFv DNA fragments wefe

amplified in second PCR reactions. Sfil and Noti sites were added to the

S'and 3'9nds of the ScFv gene. respectively.

Spun-Column" Purification: The assembled antibody ScFv gernt was

purified on a spun cofumn to remove unincorporated Iinker primas and

dNTPs.

Gel Qwntitatian of Purified ScFv Fragment: The ScFv gene fragment was

quantitated prior to IigatÎon.

Construction of intemediate plasmids

In order to get the rigM INP and c-myc expression reading frame after

cloning, pBluescrïpt II SK(+/-) and pKl8h were used as the intmeâiate

plasmids. The ScFv gene fragment was ligated into EwRV digest& ~ 8 l ~ a a i ~ t

II SK(+/-) first instead of the pCANTA83 supplied with the kit, then the neW

recombinant pfasmid was digested by BssHIf. That BssHll digea8d

fragment was finally inserted into EcoRl digested pK1 8h. generathg pK18h-myc.

The experimental procedures are described as fotlows:

a) p6luesaipt f i SK(+I-) was digested by EroRV and dephosphoryiated by

shrimp alkaline phosphatase,

b) Dephosphorylated p6luesaipt II SK(+f-) was ligated with the ScFv DNA

fragment.

c) After overnight Iigation, the DNA was transfonned into Ecoli host cell XL-1

based on the D. Hanahan method (Appendk A2) and plated on Ampicillin'

SOB culture plate.

d) The overnight cultured colonies were picked for cracking (Appendix A3)

for screening of the recombinant plasmids.

e) The picked colonies were inoculated to SOB medium and incubated

overnig ht.

f The overnight cultures were used for DNA extraction through rapid boiling

method (Hofmes and Quigley, Appendix A4).

g) The extracted DNAs were digested by Notl and Sm respectively for

orientation identification.

h) The selected recombinant plasmids were sent for sequencing.

i> The DNA sequence was compared with known cmyc antibody sequence

from NIH and named as pNScFvniyc.

j) The pNScFv-myc was digested by BssHll again.

k) pkl8h was digested by EcoRV and made blunt-ended with dNTPs.

1) Above pkl8h was then dephosphorylated by shrimp alkaline phosphatam.

m) Dephosphorylated pk18h was Iigated with the ScFv DNA fragment get

from j).

n) After ovemight Iigation, the DNA was transfmed Ïnto Ecoli host œ i l XL-1

based on the 0. Hanahan meîhod (AppendD< A2) and plated on

Kanamycin' SOB culture plate.

O) The ovemight ailturad colonies were picked for cracking (Appendk A3)

for screening of the recombinant plasmid.

p) The picked colonies were Ïnoarlated in SOB medium and incubated

ovemight.

q) The overnight cultures were used for DNA extraction through rapid boiling

method (Holmes and Quigley. Appendk A4).

r ) The extracted DNAs were doubled digested by EmRl and N d for orientation identification,

s) The selected correct recombinant plasmid was named as pki8hniyc.

3.2.4 Construction of the INPcniyc fusion protein cdi surface display vedor

p NinaZ-rn yc

The NolllEcoRI double digested 810 bp DNA fragment, Midi encoding a-

myc ScFv from above pK18h-myc. was ligated into Bsffil digested pNina2 The

experiment procedures are described as follows:

pKl8h-myc was doubled digested by NofVEmRl and pNinaZ wcro

digested by BsrGI.

The digested 810 bp DNA fragment from pK18h-myc was made blunt-

ended with dNTPs. At the sarne time, the Bsffil digested pNinaZ was

made blunt-ended and then dephosphoryiated by shnmp alkaline phosphatase.

ARer overnight ligation. the DNA was transformed into E.coli host cell XL-1

based on the O. Hanahan method (Appendix A2) and plated on Ampicillin*

SOB culture plate.

The overnight ailtured colonies were picked for cracking (Appendix A3)

for screening of the recombinant plasmids.

The picked colonies were inoailated in SOB medium and incubateci

ovemight.

The ovemight cultures were used for DNA extraction through rapid boiling

method (Holmes and Quigley, Appendix A4).

The extracted DNAs were digested by Eagl and BamHl respectiveiy for

orientation identification.

h) The selected correct recombinant plasmid was named as pNinaZ-myc

(Figure 3.5).

1 0425 bgse Unique Sites

Pvul 5686- 4757 Nhsl

m ïnaZ 81 0 bp a c-myc gene

Figure 3.5 The plasrnid rnap of pNinaZ-myc

3.2.5 Construction of the 1NPc-myc fusion protein E. coiÏ surface display vedor

- pNinaZScFv-BsaAl

The SmallEagl double digested 795 bp DNA fragment which encoding a-

myc ScFv from above pK18h-myc, was Iigated into BsaAl partial digested

pNinaZ The experirnent procedures are desuibed as follows:

pK18h-myc was doubled digested by SmaVEagl and p N i W was parüally

digested by BsaAl.

The digested 795 bp DNA fragment from pK18h-myc was made blunt-

ended with dNTPs. The BsaAl digested pNinaZ was gene cleaned by the

GENECLEAN'KIT from 810 101, Inc., Vista, CA (Appendix Al ) and

dephosphorylated by shrimp alkaline phosphatase.

After ovemight ligation, the DNA was transfomiad into E-coli host cell XL-1

based on the D. Hanahan method (Appendix A2) and plated on

Ampicillin' SOB culture plate.

The ovemight culturad colonies were picked for cracking (Appendix A3)

for screening of the recombinant plasrnid.

The picked colonies were inowlated in S08 medium and inarbated

ovemight.

The ovemight cultures were used for DNA extraction through rapid boiling

method (Holrnes and Quigley, Appendix A4).

The extracted DNAs were digested &y € d l , BamHl and Sacl

respedively for orientation identification.

h) The selected correct recombinant plasrnid was named as pNinaZScFv-

BsaAl (Figure 3.6).

2,Rll, y18m$y2 2 Ssp l 10341

2 sspa 10tn 411 AMI 2

1 CIaL 10168 437ffmI1

2 s&AI lofal 497 -12 1 EcoNI 9915 51)3 5151 2

2 PshN 9026

1 EcoW 8104 2 BssHll 8147

1 Bspl2ûi 7913 pNin;tZScFv-BsaA L 1 Apal 7943 1

1 Bcll 7750 + ' 2657 Ml3611 1 Mlul MCI 10431 base pairs 2657 Sscl 2

2 Afllll m6 Sites c= 2

2 Tthltll 7217 1 BsaU 7212

1 BstZ171 7190 2Sapl7084 38ZT 6coRl2

1 Bsp LU1 11 6962 2 AfIIII 6962

3711 Saur01 2 2 Ahdl 6û69 7 7 - 4025 PshA1 2

1 Pvul 5702 4û9ô Mt3611 2

2 Ssp 1 5267 109B Sacl 2-

-Hl 2 42ï3 SMDI 2

4755 ACCOS1 2 i 4755I in l2 4763 Acell 1 4763 - 1 1

4930 -AI 2

81 6 bp a c-myc gene

Figure 3.6 The plasmid map of pNinaZScFv-BsaAl

3.3.1 Effect of gr& medium and temperature

In order to get the optimal incubation conditions for INP and INPScFv a c-

mye fusion protein expression, different host cells and temperatures as well as

culture medium have b e n tried. They are:

Comparîson between host cells XL-1 and JM109 in SOB medium @) 37OC.

Comparison between host cells JMI09 @ 37OC in SOB and LB.

Comparison between host cells JM109 @ 22°C and 37°C in LB.

The optirniration experirnent procedure is described as follows:

An ovemight culture of EcoIi. cells was grown in LBlSOB medium. A new

inocufum was made from the ovemight culture to new medium in a l M W

dilution-

1 mM IPTG was added for induung at the early exponential growth phase

(0.4 optical density (OD)600rim )-

The culture was sampled at different times and one part was sent for OD

value measurement and the other part for ovemight plate culture.

Both samples need to be properly diluted. Viable cells were counted as

colonies plated on LBISOB plates.

3-3-2 Growth studies of E coli host ceIl XI-1, JM 109

In order to test whether suface expression of IN? and INP-ScFv a cmyc

fusion proteins inhibit the growth of host cells, E-cdi ~ells expressing INP and

INP-ScFv a c-myc were compared. The expariment procedure is same as

described in Section 3-3.1 ,

3.4 Measurement of ice-nudeation acüvity

The ice-nudeation acüvity was rneasured via Vali's method (Vaü, 1971).

The reffïgerated bath (RTE-1 10, NESLAB Instruments Inc-. NH, USA) containing

chilled reftigerant (5050 ethylene glycol and water mixîure) was preset at 4OC.

The small volumes (2Ox20pi) of diluted Ecoii solution were distributed to the

aluminum boat, then put into the bath. The nurnbers of fiozen droplets wrm,

counted on certain time intervais Always use unadivated cell solutions or celb

containing pNzanl as controls. DO water was also used to ensure the surfàœ of

the boat nucleus-free. The experïment procedure is desmbed as follows:

An 1rnM IPTG induced culture of bwli. was grown. The harvesüng tirne

was chosen at 6. 9. 12 hr respectively after IPTG activation.

1 -5 ml of culture was transferred to microcentrifuge tube and spun about 1

minute. All the supematant was removed-

The pellet was washed with 1 ml of TE three tirnes and resuspended

washed pellet into 1 ml of TE solution.

20 droplets of E.co/i TE solution were distrikited. 20vl each. on the

surface of the aluminum boat. The boat was put into 4OC bath and the

frozen numbers of droplets out of 20 were counted after 2 and 4 minutes.

In order to see whether the ice nudeation adivity is related to the su-

exposure of the INP-ScFv a cmyc fusion protein, the IPTG induced €. d i

culture which containing pNinafSbv-BsaAl was hanrested after 9 hr. The E. Coli

pellets were pretreated, with 100 pi of 1 mM EDTA/20% sucroseKris-HCICI pH8 for

30 min and 60 min @ Tr respedively, before continued to above experimental

procedure c).

3.5 SOS-PAGE and Western blottnig analysis

3.5.1 SOS-PAGE

Total lysates of cells expressing the INP and INP-ScFv a-yc fimion

protein were analyzed on SDS-PAGE and subsequent Western blotting. The

cells were grown in LB overnight and difuted 111 W into LB medium and g m at

37OC to a 0.4 OD- nin and then induced with 1mM JPTG for an additional 9 hr.

The cells were harvested. washed, and analyzed on 6% SDS-PAGE gel with a

4% stadcing gel (Laemmli, 1970). Proteins were visualized by Coommassie

brilliant blue with prestained molecular weight protein markers (Cat. 6û411At

Bethesda Research Laboratories, Life Technologies Inc., Rockville. MD).

3.5.2 Western blotting

Western blotting is also called immunoblotting, this three-step procedure is

commonly used to separate proteins and then identify a specific protein of

interest.

The protein ample is subjected to SDS-PAGE gel nrnning first After the

gel ninning, a nitrocellulose membrane is tightly applied to the face of the gel.

Then an electric field is applied. Protein bands Ri the gel are transferred, also called &lot to the membrane. Nitrocellulose membrane is a substance that

strongly and nonspecifically binds proteins. Nylon or polyvinylidene-difiuonde

also can be used. In the second step, the membrane is soaked in a solution of an

antibody (Ab,) speafic for the protein of interest. The excess adsorption sites on

the nitrocellulose membrane are pre-blocked with nonspeafic proteins such as

casein, skimmed milk to prevent the nonspecific adsoiption of the antibodies. In

the final step, after washing away the unbaind A h , the block was incubated with

an enzyme-linked antibody (Ab) to identify the band Wntaining the protain of

interest,

In this study, nitrocellulose membranes were used for blotüng (Appandir

AS). Blotted membranes were piaœd in a blocking solution of 5% skim milk in

PBS bmer for 90 min. For immunodetection. Anti-Mouse IgG (FAB Specific) was used as A b since expressed ScFv a cinyc played the role as Ab,. This A b was

covalently Iinked to Alkaline Phosphatase Conjugate (Produd No. A1293. Sigma

Co.. St Louis, MO), which was diluted in PBS (t5000). The color reactim of

alkaline phosphatase was according to the manufacturer's instrucüons (Sigma

Co., St. Louis, MO)(Appendix A6).

3-6 Flow cytometry

3.6.1 lmmunostaining of the bacteria

a) Ecdi cells without pretreatment

E . d culture was grown in LB @ 37OC and induced with IPTG as 00-

reached 0.4. After 9 hr culture, 1 ml of culture was harvested and then washed

by 1 ml of PBS for 3 times. The E. mli pellet was resuspended in 1 ml of PBS

with 3% skimmed milk for 20 minutes blocking then incubated with 40 pi uf FiTc

linked 2nd antibody (Fluorescein (FiTC)oonjugated Affinif ure Goat Anti-Mouse

IgG (H+L), Jackson immunoResearch Laboratories. Inc.. West Grove, PA) for 1

hour on ice. After washing with 1 ml of PBS @Tr for 5 times. the E-cdi was

analyzed by flow cytometry (FACScan, Becton Dickinson, Oxnard, CA).

b) E-mli cells with EDTA pretreatrnent @O°C on ice

E-coli cultures was grown and induced with IPTG as 00-rn reached 0.4.

After 9 hr culture, 1 ml of culture was harvested and washed by 1 ml of PBS. The

pellet was treated with 100 pl of imM EDTAI20% sucroseKris-HCI pH8 for 30

min @ 0°C on ice and washed once with 1 ml TBS/250 mM sucrose/lO mM

MgCl* @4OC. The E- cdi pellet was resuspended in 1 Wpl of PBSnJOmM

sucrose/l O mM MgCI2. Then incubated with 1 -5 pl fluorescent c-myc peptide for

30 min @ 37OC. After washing with 1 ml of TBSf250 mM suuose/lO mM MgC12

@4OC for 5 times. the E-coli was anal- by fiow cytometry (FACScan, Becton

Dickinson. Oxnard, CA).

c) E-coli cells with EDTA pretreatment @Tr

E-wli culture was grown and induced with IPTG as ODmm reached 0.4.

After 9 hr culture, 1 ml of culture was harvested and washed by 1 ml of PBS. The

pellet was treated with 100 pl of I m M EDTA120% su~~~seKns-HCl pH8 for 10

min @ T, and washed once wïth 1 ml TBS1250 rnM sumse/lO rnM MgCh W0C.

The E. mli pellet was resuspended in 100pl of PBSn50mM suaosdl0 mM

MgCl** Then incubated with 1.5 pI fluorescent c-myc peptide for 30 min @ 37OC.

After washing with 1 ml of T B S B mM sucroseil0 mM MgC12 @4OC for 5 times,

the Ecoli was analyzed by flow cytometry (FACScan, Becton Dickinson, Oxnard. CA). As a control. unstained cells were used for flow cytometry.

3.6.2 Preparation of fluorescent antigen

The c-myc-peptide CGAEEQKLISEEDU was coupled to

fluoresceinmaleimide (Pierce, Rockford. IL) according to the instruction of the

manufacturer. Uncoupled dye was separated by G1O Sephadex (Pharmacia,

Piscataway, NJ) on chromatographic column. The experiment procedure is

descri bed as follows:

1 mg of c-myc peptide was dissolved in 1 ml of PBSl15O mM NaCl pH79

with 1 rnM EDTA.

150 pi of peptide was removed into 1.25 ml of PBS with 1 mM EDTA

100 pi of DMF with 4 mg of Fluorescein-5-Maleimide was added into

above PBS solution,

Then the mixture was incubated in the da& @Z°C for 2 hr.

80 mM P-mercaptoethanol was added to quench the reaction.

The mixture was nin through a G1O Sephadex and the filtrate was stored

@ -20°C until next use. The c-myc peptide concentration is -1 nrnoV1OpI.

Chapter 4 Results and Discussion

4-1 Introduction

The results of the expenments perfomed are discussed in sequence with

the order of experimental methods desaibed in Chapter 3. flot necessary in the

order they were performed.

4.2 Construction of surface dispiay vectors

4.2.1 Construction of the INP coli surface display vector - ~NinaZ

Based on the results of cracking, three colonies, nameiy 629, Cl5 and

Di6, were picked up for ovemight culture and then their plasmid DNAs Mwe

extracted through rapid boiiing method. The extracted plasmid DNA were

digested by BssHII for orientation identification-

According to the DNA recombinant plasmid prediction. the BssHll

digestion results should be:

BssHI I 2 fragments if the orientation of insertion is 5-3'

Order Length From To

1 6544 787 BssHll + 7331 BssHll

2 3071 7331 BssHII + 787 BssHll

BssH l l 2 fragments if the orientation of insertion is 3'4'

Order Length

1 5249

From To

7331 BssHll + 2965 BssHll

However. the digestion result showed that 629 was divided into thne

fragments instead of two as:

Order Length From To

1 -1000 787 BssHll + -1 787 BssHII

2 -5000 -1 787 BssHl l -+ 7331 BssHll

3 3071 733 1 BssHI 1 + 787 BssHll

The digestion results showed that D16' and Cl 5 were also divided into three fragments as:

Order Length From To

1 -5000 2965 BssHl l + -3965 BssHII

2 -3366 - 3965 BssHII + 7331 BssHlf

3 5249 7331 BssHll + 2965 BssHll

Those results showed that there is an unknown BssHll cutting site on the

DNA fragment from pUC18131CE. That unexpected site could be caused by an

incidental deoxyribose nucleotide replacement However it did not affect the

reading that 829 is in the 5'-3'. the right orientation and both Cl 5 and Dl6 are in

the 3'-5' orientation. 829 and Dl6 were named as pNinaZ and pNzani

respectively.

pNinaZ was sent for sequencing and results confirmed that it contains the

right 3600 bp inaZ gene after being compared with sequence in GenBank.

The predicted five unique cutting sites digestion results showed that 437

Hpal is not a unique site on pNinaZ which could be caused by an incidental

deoxyribose nucleotide replacement in pSD. So. there are 503 Sm, 1634 Aafll.

2657 Sacl and 3260 BsrGI, available for the ScFv a c-myc gene insertion.

Among above unique sites. Sfil is in the unique N-terminal region, from 140 to

665, which is not ideal for insertion and the other three are well in the central

repeating zone. Considering that BsrGf is the one which is nearest to the C-

terminai, BsGl was chosen as our insertion site for the centrai repeating zone.

4.2.2. Preparation of DNA fragment encoding ScFv as-myc

Instructions of ~ u i c k ~ r e p ~ mRNA Purification Kit (XY-039-00-07, rev.4)

and Instructions of Mouse ScFv ModuleIRecombinant Phage Antibody System

(XY-025-00-10, revJ) from Pharmacia Biotech were followed step by step during

the whole preparation. A gel quantitation of purified ScFv fragment was nin on

1.5 % agarose gel (Figure 4.1). Compare the intensities of the bands in the lanes

containing assernbled product (Lane 6. 7) with the ScFv Marker band (Lane 4, 5)

that correspond to the size of the assembled product (-750 bp). This marker

bank contains approximately 12.5 ng in the 2.5 pl aliquot (Lane 4) and

approximately 25 ng in the 5pl aliquot (Lane 5). It could be seen that the intensity

of 4 pl assembled product (Lane 7) is much greater than that of the 12.5 ng

marker band (Lane 4) which means the estimate volume of the assembled

product is corresponding to 0.25 -1pg- lt is high enough with this concentration

for the succeed restriction digestions.

Figure 4.1 Gel Quantitation of Purified ScFv Fragment

4.2.3 Construction of intemediate piasmids

Based on the results of first terni cracking (Section 3.2.3 d)), fÏve colonies,

namely A4. 6, 9, 10. 19. were picked up for ovemight culture and then their

plasrnid DNAs were extracted thmugh rapid boiling method. The extracteci

plasrnids DNAs were digested by N d and SlRl respectively for orientation

identification.

According to the DNA plasmid prediction. bath Non and Sffll digestion

results should be two fragments if the orientation of insertion is 5-3'.

The results showed only A4 and A9 met the requirements. They were sent

for sequencing and results confimed that both of them containing the rigM 750

bp ScFv a cmyc gene after compared with known c-myc sequence from NIH.

The A4 was named as pNScFv-myc.

Based on the results of second terni cracking (Section 3.2.3 O)). five

colonies. namely Al, 2. 8. 65. 14. were picked up for ovemight culture and then

their plasmid DNA were extracted thmgh rapid boiiing method. The extraded

plasmid DNA were double digested by Noll and EcoRll respectively for the

confirmation of there is a 810 bp DNA fragment which contains the right 750 bp

ScFv a c-myc gene. The restriction enzymes chosen here are in order to keep

the right reading frame of both INP and ScFv a c-myc after cloning. This is why

the DNA fragment finally inserted into pNinaZ is not 750 bp but 81 0 bp instead.

Only A8 was show to be the correct recombinant plasmid. It was named

as pK1 8h-myc. For pK1 8h-myc there was no orientation problern as that 810 bp

DNA fragment was digested again and ligated into pNinaZ.

4.2.4 Construction of the 1NP-c-rnyc fusion protein E culi surface display vector

pN inaZ-myc

Based on the results of cracking. totally 33 colonies, namely 3, 12. 17, 18,

24, 38, 42, 45, 47, 50, 54, 73, 79. 85. 88. 89, 90, 102, 107, I l 0, 1 1 1, 11 7, 126,

131. 132. 135. 140. 166. 170, 148. 177, 181 and 184, were picked up for

overnight culture and then their piasmid DNAs were extracted through rapid

boiling method. The extracted plasmid DNAs were digested by Eagl and BamHl

respectively for orientation identification.

According to the DNA recombinant plasmid prediction, the Eagl digestion

results were:

a) Eagl 6 fragments if the orientation of insertion is 5-3'

Order Lerqth From To

1 168 1708 Eagl + 1876 Eagl

2 984 1876 Eagl + 2860 Eagl

3 72 2860 Eagl -+ 2932 Eagl

4 1134 2932 Eagl + 4066 Eagl

5 5531 4066 EagI + 9597 Eagi

6 2536 9597 Eagl + 1708 Eagl

In order to compensate the sire gap of 1 kb commercial ladder for easy

recognition, pNinaZ was digested by Eagl at the same time. According to the

DNA recombinant plasmid prediction, the Eagl digestion results on pNinaZ were:

b) Eagl 5 fragments

Order Length

1 168

2 984

3 72

4 5855

5 2536

From To

1708 Eagl + 1876 Eagl

1876 Eagl + 2860 Eagl

2860 Eagl + 2932 Eagl

2932 Eagl + 8787 Eagl

8787 Eagl + 1708 Eagl

The actuai Eagl digestion results showed there were only three bands for

pNinaZ and maximum four bands for the possible recombinant plasmids on the

gel. It is very possible that the small DNA bands may have dense problem to be

visible on the 1.2% agarose gel with 1 kb ladder.

However. colony No. 3, 50. 135. 140. 166, 170 had shown four bands

with the proper size compared to digested pNinaZ In order to make sure that

they were the correct recombinant plasmids. their DNAs were digested by BarnHi

again.

According to the DNA recombinant plasmid prediction, the BarnHf

digestion resufts were:

b) BamHl 2 fragments if the orientation of insertion is 5-3'

Order Length From To

1 3877 26 BamHl -+ 3903 BamHl

2 6548 3903 BamHl + 26 BamHI

Colony 3, 50, 140, 166, 170 showed the expected digestion results.

Combined with the results from above Eagl digestion. It was known know that

colony 3, 50. 140. 166 and 170 are the nght recombinant plasmids. Colony 170

was picked up randomiy and named as pNinaïniyc.

4.2.5 Construction of the INP-c-myc fusion protein E. coli sufface display vector

- pN inaZScFv-BsaA l

Based on the results of cracking, 14 colonies, namely 6, 14, 15, 23, 26,

37, 55, 61, 77, 74, 76, 99, 127, 146 were picked up for ovemight culture and then

their plasmid DNAs were extracted thrwgh rapid boiling rnethod. The extraded

plasrnid DNAs were digested by EmRI, BamHl and Sacl respectively for

orientation identification,

According to the DNA recombinant plasmid prediction. the EcoRI, BamHl

and Sacl digestion results were:

a) EcoRl 2 fragments if the orientation of insertion is 5-3' and the

insertion is in site 361 8 not 641 7-

Order Length Frorn To

1 6802 3627 EcoRl + 10429 EcoRl

2 3629 1 0429 EcoRl + 3627 EcoRI

b ) BamHI 2 fragments if the orientation of insertion is 5-3' and the

insertion is in site 3618 not 6417.

Order Length From To

1 6191 26 BamHl + 4266 BamHI

2 4240 4266 BarnHf + 26 BamHl

C) Sacl 2 fragments if the orientation of insertion is 9-3' and the insertion is

in site 361 8 not 641 7,

Order Length From To

1 ? 441 2657 Sacl + 4098 Sacl

2 8990 4098 Sacl + 2657 Sacl

Onfy colony No. 6. 14, 15 met al1 above predided results. They are the

correct recombinant plasmids. Colony 14 was picked up randomly and narned as

pNinaZScFv-BsaAl.

4.3 Growth studies of E, coli cell- Xi-1, JM109

4.3.1 Effect of growîh medium and temperature

1) Cell growth cornparison between host cells XL-1 and JMlO9 in SOB

medium @ 37OC.

The plasmids pNinaZ and pNRiazmyc were introduced into E. di host

cells XL-1 and JM1O9 respectively and incubated in SOB @ 37OC for their growth

observation. From the resuits Figure 4.2. we could see that the growth rate of JM

109 is much faster than XL-1 both for the cells containing plasmid pNineZ and

pNinaZ-myc. Also h m the resuits of their viable ceIl numbers, Figure 4.3. it

could be easy found that JM109 is a more efficient host cell than XL-1 for the cell

growth.

t ima (hr)

Figure 4.2 Cell growth of XL-1 and JM 109 in SOB @ 37O C

Tirii. (hr)

- -

Figure 4.3 Viable ceil nurnbers of XL-1 and JM 109 in SOB @ 37O C

There were initially problems with JM109 cells incubation @ 37OC. The

cells were incubated in test tubes on rotary shaker. The ceils lysed gradually

after IPTG activation. The OD mm value of cells containing pNinaZ-myc would

drop to as low as 0.30 and 0.1 3 after 3 and 6 hours respectively (Figure 4.4). The

ODm nm values could be gradually increased after 9 hours. The problem was

finally solved by incubated the cultures in baffled flask under vigorous s W n g

aeration. Comparing Figure 4.3 with Figure 4.4, it was found as the IPTG were added in at the early stage of ceil's exponential phase. cells were at their fastest

propagating rate and it's iikely that dissolved oxygen may have becorne the

critical factor lirniting growth.

Figure 4.4 Cell growth of JM 1 09 in SOB @37 OC in rotating shaker (rprn 1 5 )

2) Cell growth camparison of host cell JMI09 @ 37°C in SOB and LB

JM109 ceils containing the pfasrnid pNinaZ and pNinaZ-myc were

incubated in SOB and LB @ 37OC for their growths. From the results Figure 4.5

and Figure 4.6, it was seen that the growth rates of JM 109 cells in LB and SOB

were not signifcantly different It even seerned that SOB is a more suitable

medium than LB for cell growth. However, considering both INP and S b v a c-

myc fusion proteins have been expressed in LB medium by other researdlers

(Jung et al., 1997(a), 1997(b); Fuchs et al.. 1996), LB was finally chosen as ouf

culture medium.

Figure 4.5 Cell growth cornparison of JM109 @ 37°C in SOB and LB

Figure 4.6 Viable cell numben of JM 109 in SOB and 16 @ 37' C

3) Effect of INP growth of host cells JM109 in LB @ 22°C and 37OC

Before the autolysis problem of JM109 inaibated @ 37OC was solved,

incubation of cells under lower temperature @ 22°C was carried out. This was in

order to reduce the growth rate of cells to avoid the autolyolysis and also to

irnprove the fusion protein expression. Gurian-Sherman and Lindow (1995)

reported that large differences in ice nucleation frequency occurred at al1 but the

lowest assay temperatures in cells of Psuedomanas syringae grown in the

temperature range of 15 to 33 OC. That differences in ice nudeation frequency

may be attributed to differences in the total nurnber of nudei present. which is the

INP, in the population of cells. As the inaZ gene was originally from

Psuedomanas symgae, it was necessary to verify whether it applied to

recombinant E-coli Though the growth rate of JM109 @ 22% was really slow

wmpared to Q 37OC (Figure 4.7), the expression of INP fusion protein could be

largely improved anyhow.

However the SOS analysis showed no evidence to support this trial

(Figure 4.8). After the auto-lyolysis problem of JM109 inwbated @ 3i0C was

solved, no further temperature studies were required-

Figure 4.7 Cell growth cornparison of JM109 in LB @ 22°C and 37OC

ScFv

INP .

Figure 4.8 SDS result of INP and INP a ScFv c-myc expression

@37OC and 22°C

Lane land 2 are the XL-1 host cells expressing plasmid pNinaZ-myc and

pNinaZ incubated 9 hr after IPTG induction @37 OC. Lane 3, 4 are XL-1 host

cells expressing plasmid pNinaZ-myc and pNinaZ incubated 9 hr after IPTG

induction @22 O C then put @ 4 OC for ovemight. No significant differences were

obsewed behrveen Lane 1 and Lane 3, lane 2 and Lane 4-

4.3.2 Growth studies of host celIs JM 109 cuntaining pNinaZ pNiMZ-myc and

pNinaZScFv-BsaA l

In order to test whether surface expression of INP and [NP-ScFv a cinyc

fusion proteins inhibit the growth of host celis, the growth of EcolJ cells

expressing pN inaZ, pNinaZ-myc and pNinaZScFv-BsaAl were cornpared with the

cel! expressing pSD. No obvious inhibitions were observed during the stationary

phase (Figure 4.9 and Figure 4.10).

T ime (hr)

Figure 4.9 Cornparison among JM109 containing pSD, pNRia2, pNlnaZniyc

and pNinaZScFv-BsaAl in SOB @ 37OC

O 3 6 9 12 S 18 21 24 21 30 33 36 39 4 2 45 48

Tirne (hr)

Figure 4.10 Viable cell numbers of JM109 containing pSD, pNina2, pNlnaZ-

myc and pNinaZScFv-BsaAl in SOB @ 37%

4.4 Measurement of ice-nucleation adivity

Ice-nucleation activities suppfied an additional measurement for verWying

whether the fusion protein is functionally expressad and has its biological

conformation on the surface of Elwlii Hawever the results showed that whether

there is an ice-nucleation activity does not necessanly relate the integntty of the

INP. lnstead the ice-nudeation activity is more related to protein's tefüary

structure. The results showed on Figure 4.1 1 a) and b) confonn to the other

researchers' (Green a t el.. 1 988).

As mentioned in 4.2.1, pNznal has the 3-5' wrong orientation of that 3600

bp inaZ gene insertion. It has the same site as pNinaZ but without INP

expression function. This charader makes it an ideal control plasmid. pNinaZ ' s

ice nucleation activities indicated that it is a well constructed !NP surface

overexpression plasmid. The interesting point is the cornparison between

pNinaZ-myc and pNinaZScFv-BsaAl. Both of them are INPScFv a c-myc fusion

protein expression plasmids. However the ScFv gene was inserted at different

regions of that 3600 bp inaZ gene. As mentioned in 4.2.4. for pNinaZ-myc, the

ScFv gene was inserted in the central repeating zone. The ice rtucleation

activities were reduced only slightly. For pNinaZScFv-BsaAl, the ScFv gene was

inserted in the unique C-terminal domain. The cutting site is only 20 amino acid

away from the end of C-terminal and Class I ice nucleation activities were also totally eliminated-

It seems that EDTA pretreatment did not increase the ice nudeation

activities of E. coli which expressing pNinaZScFv-BsaAl a lot. A slight increase

was observed. However, that increase could be caused by chance. The ~~SLJ I~S

suggest that the ice nudeation acüvity is more related to the structure of the

fusion protein itself than the protein surface exposure degree.

_ _A.- _-- _ - ___ _-- ___---- DpNtani mpN-lZ - - O~N~!I~Z-KIYC OpN&aZScFy-BsaAI - --mEDTA 30 min OEDTA 60 m i n -" - . ---- -- - - - - - - -- - --

6 S Time (hr)

Figure 4.1 1 a) INP activities of JM 1 09 in LB @37Oc counted after 2 minutes

b) INP activities of JM1 09 in LB @37Oc counted after 4 minutes

4.5 SDS-PAGE and Western blotting analysis

4.5.1 SDS-PAGE

The SDS-PAGE gel showed that the INP-ScFv a c-myc fusion proteins

expressed by different recombinant plasrnids, pNinaZ-myc and pNinaZScFv-

BsaAl shared the same molecular weight (Figure 4.12). The MW of INP is

approximately 160 KD and INP-ScFv a c-myc fusion proteins are approximately

190 KD which is in confomity with the prediction. as the MW of ScFv a C-mye is

30 KD (Fuchs et al., 1997).

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Marker pNinaZScFv-BsaAl pNinaZ-myc pNinaZ pSD

Figure 4.1 2 6% SDS-PAGE

4.5.2 Westem blotting

A lot of efforts were put into Western blotting. Different blocking agents,

Ab2 and incubation times were tried. Unfortunately the results were not

satisfactory as expected. Several problems are 1 isted below;

The first problem was the blotting. As mentioned in Section 4-51, the INP-

ScFv a c-myc fusion protein is approximately 190 k0 which is quite large size

compared to other Ewii proteins. The blotting is becoming vefy difficult due to its

large size. In the expariment. the blotting condition has been increased from

normally 200-250 mA for 1 hour to 850 mA 11 OOV for 2-2.5 hours in order to Mot

the protein from SOS-PAGE gel to the membrane. Temperature needs to be

carefull y controlled-

Blocking was another problem which needs to be solved. Different

blocking agent such as 3% BSA. 3% casein, 2% gelatin and 5% skimrned milk

were used. Also different blocking times have been tried. The results were not

satisfied. Either there was no band on the membrane showed up due to

overblocking (e-g. blocking ovemigM @ Tr) or every bands showed up on the

membrane after color deveiopment due to not enough blocking (e-g blocking 4 hr

@ Tr).

The optimal conditions for the Western blotting anaiysis of INP-S~FV a c-

myc fusion protein is still need to be investigated. As later shown in the flow

cytometry results in Section 4.6.1, Ano-Mouse IgG is easy to be contaminated

then cause nonspecific binding to E. d i proteins. The c-myc peptide-AP is

suggested to instead of Anti-Mouse IgG -AP as the A b i n future study.

4.6 Flow cytometry

4.6.1 lrnrnunostaining of the bacteria

a) Ecoii cells without EDTA pretreatment

As described in Section 3-63 a), initially flow cytometry of untreated

cells was canied out

Figure 4.1 3 shows the flow cytometrical analysis of E. coli cells expressing

plasmids pSD. pNinaZ and pNinaZ-myc. The upper row (a), (b) and (c) showed

the auto-fluorescent peaks observecl M e n no FITC-anti Mouse lgG (H+L) is

added. Addition of FITC-anti Mouse IgG (H+L) causes a shiR in the fluorescent

counts. These shifts were caused by the binding of FITCanti E. coli IgG which

are contamination of the FiTCanti Mouse IgG (H+L). In our case, fumer skrdies

were carrÏed out with fluoresceinmaleimide (FM)-c-myc peptide as described in

Section 3-62.

Only a very small peak was observed in (c') of bottom row, which stands

for E. coli cells expressing pNinaZ-myc. That peak is pointed by an a m . It seems that while the expression of tiny part INP-ScFv a c-myc fusion proteins

pierdng the outer membrane and makes them accessible of antigen binding,

others retained in the periplasmic space-

a 3

no antibodies

added

Fluorescence intensity

Figure 4.1 3 Flow cytometrical analysis of E-coli expressing pSD. pNina2,

pNinaZ-myc that are stained by FITC-anti Mouse IgG (H+L)

b) E . W cells with EDTA pretreatment @O°C on ice

TO overwrne antigen binding problem, EDTA was used to inuease the

permeability of the bacterial outer membrane (Leive, 1968). And in 0- t0

irnprove the specificity of the antigen, fl uoresceinmaleimide (FM)*-myc p-de

was used instead of the F TTC-anti Mouse IgG (H+L).

Figure 4.1 4 shows the flow cytometrical anal ysis of E. cdi ceils expressing

plasmids pNinaZ and pNinaZ-myc. The upper row (a), (b) showed the auto-

fluorescent peaks observecl when no F Mc-mye peptide is added.

There was a quite large peak seen in (b') of bottom row, whicb stands for

E. coli ceils expressing pNinaZ-myc (see arraw indexed point). No fluorescent

shift caused by non-speàfic binding was observed. The results show that the

antigen binding situation has been Rnproved due ta EDTA membrane treatmemt

+ no FM.C-myc

added

FMcniyc

peptide

Fluorescence intensity

Figure 4- 14 Flow cytometrical analysis of Ecoii expressing pNinaZ. pNinaZ-

myc stained by fluorescent c-myc peptide and treated by EDTA @O°C on ice

c) E-mti cells with EDTA pretreatment @T,

In order to further increase the pemeability of the bacterial Outer

membrane, the EDTA pretreatment condition was modified. The inabaiion

temperature was changed from O°C to room temperature. The incubation time

was shortened from 30 min. ta 1 O min,

Figure 4.15 shows the flow cytometricaf analysis of coli cells expressing

plasmids pNinaZ pNinaZ-myc and pNinaZScFv-BsaAl. The upper row (a), (b)

and (c) showed the auto-fluorescent peaks observed when no F Mc-myc peptide

is added. A great improvement of antigen binding in (b') of bottom row, which is

the ceIl expressing pNinaZ-rnyc, was observe& That peak is pointed by an index

finger in the figure-

no FM<-myc

added

4- FM<-WC peptide

Fluoresœnce intensity

Figure 4.1 5 Flow cytometricaf analysis of Ecdi expressing pNina2, pNinaZ-

myc and pNinaZScFv-BsaAl stained by FM<-myc peptide and treated by EDTA

@Tr

At the same time, a fluorescent shift caused by c-myc antigen binding was

observed h (c') of bottom row which means that with EDTA treatment a part of

the fusion protein expressed by pNinaZScFv-BsaAl was also exposed to the

surface of E: coii-

It is very interesting !O compare the results between (b') and (c') in Figure

4.15, which stand for the Ecdi cells expressing pNinaZnyc and pNinaZScFv-

BsaAl respective1 y. pNinaZScFv-6saAl was construded after the results from

pNinaZ-myc were obtained. The insertion site of ScFv gene was only 20 amino

acid away from the C-terminal of INP. It was hypothesized that as the C-terminal

is more hydrophilic than the central repeating zone. the INP-ScFv a cmyc hrsion

protein expressed by pNinaZScFv-BsaAl should increase expression to outer

membrane surface cornpared to pNinaZ-myc's. In other words, it would have a

better surface antigen binding afinity. Hwever, the FACS results did not show

significant improvement. Fuchs et al. (1 997) expressed the ScFv a c-myc on E.

coli membrane by means of PAL motif.. They also needed to cany out

membrane pretreatment in order to observe the binding with c-rnyc peptide

antigen. It is likely that this partiwlar ScFv may have problems being transpart

to the outer surface of membrane. On the other hand, as mentioned in Section

2.2.4, the sewndacy and tertiary structures of INP are still unknown. It is premature to make the conclusion in this study. Fumer investigations are

needed for a reasonabfe explanation of above phenomena. Some suggestions

are rewmmended in Chapter 5.

Chapter S Conclusions and Recommendations

In this study, a fusion protein consisting of the INP from P. sythgae and

ScFv a c-myc was expressed on the membrane of E. cdi JM109. This

observation is based on the foilowing facts:

1 ) The E mii ceils expressing pNinaZ-myc plasmids retained ice nudeatîm

activity. This indicates the fusion protein was in the outer membrane of the

bacteria. The E. coli ceils expressing pNinaZScFv-BsaAl was construded by

inserting the ScFv encoding gene in the C-terminal of the haï . Previous studies

have suggested a loss of Class I INA (Green et al.. 1988). This was also

obsenred. However, the SOS-P AG€ showed identical protein indicating thb

protein is also in the membrane as some INA was still observed.

2) The FACS studies showed that specific binding of the ScFv ta the peptide

epitope of the c-myc antigen was observed in E. cdi cells which were treated by

EDTA for increasing the permeability of the bacteriai outer membrane. This

indicated that the ScFv portion of the fusion protein still resided inside the outer

membrane. Utilizing the clone expressing pNinaZScFv-BsaAl where the ScFv

portion is closer to the hydrophilic C-terminal region did not result in any

significant improvement. This has also been shown by Fuchs et al. 1997,

expressing ScFv ac-myc on Eco/i by PAL motif, who also needed to carry out

membrane pretreatment to observe the c-myc peptide - acinyc ScFv binding.

Thus it is possible that the particular ScFv for cniyc may have probiems being

transported to outside the cell and further optimization studies are required.

In this study, it has been demonstrated that a fusion protein INP-ScFv a c-

myc is located in the outer membrane of E. coli. However, as an E. coli surfaœ

expression motif. INP needs much more investigation to understand its

mechanism. Following suggestions are recommended to the further study.

1 ) lnsert ScFv a c-myc gene into the unique C-teminal region of inaZ again.

The position should be just before the C-terminal stopping codon. As the

C-teminal unique domain rernains integcity, ice nudeation actïvity could

expect to be retained. It rnay also improve the surfaœ pemeability.

2) lnsert ScFv a c-myc genes into the repeating central region of inaZ again.

Compare their surface antigen binding abilities to their insertion position. It

rnay help to understand the tertiary structure of INP.

3) Explore the maximum transportation ability of INP, knowing what is the

largest size of protein that can be transported by the system. And trying to

express two foreign proteins in an INP- A -B fusion mode[.

4) Extraction of INP-ScFv a c-myc from its outer membrane and its

application could be considered. Of course, M o l e cell immobilizatim"

wiil be an alternative after the surface pemeability has been further

irnproved.

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Appendix A Experimental Protocols

Appendix A l GENECLEAN@ (Short Proto~olMBIO 101 Inc., Vista, CA)

When DNA is wntained in agarose, indude the parenthetical statements.

1. (Excise band from agarose gel.)

2. Add 3 volumes of Naf stock sofution. (Incubate 5 minutes at 45OC to 5S°C

dissolve agarose.)

3. ACM GLASSMILP suspension. incutmte for s minutes.

4. Pellet GLASSMILK? IDNA cornplex for 5 seconds. Remove supernatant

and set aside,

5. Wash pellet 3 times with NEW WASH.

6. Eiute DNA into water or low-sait buffer.

The above procedure takes about 15 - 20 min. to complete.

Appendix A2 Transformation of E. coli(D. Hanahan)

Reagents, Equipment

200 J Competent E.mji cells (in micro tubes in -70°C freezer)- 1 per sample

plus 1 each for negative and positive controls.

Ice in ice bucket,

42'C water bath- 37OC water bath-

S.0.C. culture medium-

S.O.C. medium is identical to SOB, except that it contains 20 mM glucose

Empty sterile 15 ml tubes.

Culture plates containing appropriate antbiotics-

Method

Clean bench working area with 70% ethanol. Thaw competent cells at r o m

temperature until the cell suspension is just Iiquid. Plaœ in ice bucket.

Add DNA mixture, 3-5 pi.

Swirl to mix,

lnwbate on ice for 30 min.

Heat shock the cells by placïng in 42°C water bath for 90 seconds.

Retum the tubes immediately to ice bucket.

Add 800 pi S.O.C. culture medium (with sterile tip) and transfer al1 of the

solution to a steriie empty culture tube.

Shake the tubes in a 37OC water bath for 3040 min.

Plate 100 pl on culture plates. Incubate ovemight at 37OC.

Appendix A3 Cracking method

2X STOCK

Tris-HCI pH 7.8

EOTA

SOS

Sucrose

bromophenol blue

H20

Bacteria preparation

Pick potential plasrnids off the transformation plate and make a patch of about 1

cm square on a seledive plate. Grow this at 37OC for about 12-18 hours, 4872

hours at room temperature also works. If desired. the plates can be stored et 4OC

for several days.

Method

Scrape the patch (-0.5 cm square) off the plate and resuspend in 100 pl d 1 O +l

TE buffer. Add 100 pi of 2X buffer, mix. incubate at room temperature for 10 -20

minutes and centrifuge in an eppendorf centrifuge for 15 minutes. Load the non-

viswus supernatant only (usually < 25 pl). (One can also add 10 -20 pl of R W e

A to get rid of the most of the small RNA. This step is necessary to observe

plasmids c 3 4 kbp in size.

91

Appendix A4 Rapid boiiing mahod for plirsmid DNA (Holmcw and

Quig iey)

Grow an ovemight culture of bacteria.

Transfer 1.5 ml to microcentrifuge tube and spin about 1 minute. Remove al1

the supematant.

Add 250 pl of STET buffer: 8% sucrose, 5% Triton X-100, 50 mM EDTA, 50

mM Tris pH 8.0. Vortex or pipette to resuspend cells completely.

Add 10-25 pl of 10 rnglml lysozyme, mix. Incubate at room temperature 10

minutes.

Boii 60-90 sec.

Centrifuge 10 min. Transfer supematant to new tube, and add an equal volume of isopropanol.

Mix. Centrifuge I O min-

Wash pellet once with 150 BI of TE. The DNA solution is frothy from the Triton

and denatured protein. Use 2 pi for the standard sized (20 pl) restndion

diaest, Add 1 ul RNAse A to each restriction diaest.

Appendix AS Western blotüng

Run SOS-PAGE gel as usual. Remove stacknig gel, notch one m e r of

the resolving. When using the Ounn carbonate bmer systems for transfer,

the gel need not to be pre-equilibrated.

Recipe of Dunn carbonate buffer:

Methanol 400 ml

Distilled Water 1600 ml

Sodium Hydrogen Carbonate (NaHCO3 ) 1.7 gm

Sodium Carbonate (Na2C03 ) 0.64 gm

Cut nitrocellulose to exact size of gel. Wet one side of nlc in transfw buffer

and when the first side is hydrated, wet the other side. Keep wet until

ready to use-

Cut 4 pieœs of 3MM paper ta 8x10 cm; wet in trander buffer. Wet the

brïllo pads in buffer so that there are no air bubbles trapped.

Assemble the above pieces on the GREY side of the holder in the

following order and ensuring that no air bubbles are trapped between the

1 ayers: GREY: Brillo pad-3MM paper-gel-nio3MM paper4ril lo pad:CLEAR

Shut clamp.

FiII the resewoir tank to 2/3 to 3/ 4 full of cold transfer buîTer and lower the

blot sandwich into the transfer assembly making sure that no air bubbles

get trapped. Fill the tank with transfer buffer, add a magnetic stir bar and

put the wver on. Plaœ the entire assembly tank into an ice bath on top of

the stir plate and make sure stimng occurs.

f ) Transfer 2 hour el00 V constant voltage. It may necessary to change the

ice in the ice bath once for keeping the temperature of buffer remains

below 3S°C.

Appendix A6 Immunodetection

Remove the blots and place in a small plastic box containing 1 Oml of Blot

Rinse Buffer containing 3% BSA and 5% skimmed milk respectively for 2

hr @ T, for blocking.

Recipe of 1 OX Hot rime buffer (final pH 1-58.0):

1 M Tris-HCI. pH 7.4

Sodium Chloride

0-1 M EDTA, pH 7-5

Tween 20

Sodium Aride

Distilled Water

For immunodetection, Anti-Mouse IgG (FAB Specific) Alkaline

Phosphatase Conjugate (Product No. A1293, Sigma) was diluted in Blot

Rinse BuRer (1 :5000) and inwbated @ T, for 2 hr.

lmmunoblots were rinsed three times with Blot Rinse Buffer, each time 20

minutes to ensure removal of unbound antibody prior to color

development.

Develop blot: making detection solution

Recipe of Develop blot buffer:

50 mM tris-HCI pH 8-01 100 mM NaCl

a-naphtyl phosphate

Fast Blue RR

e) Drain nk, rinse with little 50 mM Tris11 00 mM NaCI. Drain and pour on

developing solution. shaking. bands could be appeared immediately.

Leave up to 15 minutes with shaknig.

f) Stop with water for a few min. Then nnse in 1 % acetic acid for 5 min. And

rinse again with water.