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Johnson and B S CoopermanMcLarney, N Naidoo, O L Schoenberger, J LH Rubin, Z M Wang, E B Nickbarg, S1-antichymotrypsins.and variant human alphabiological activity of recombinant nativeCloning, expression, purification, and:

1990, 265:1199-1207.J. Biol. Chem.

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THE JOURNAL OP BIOLOGICAL. CHEMISTRY

0 1990 by The American Society for Biochemistry and Molecula r Biolog y, Inc.

Vol. 265, No. 2, Issue of January 15, pp. 1199-1207,1 99O

Pr in ted i n U .S . A .

Cloning, Expression, Purification, and Biolog ical Activity of

Recombinant Native and Variant Human arl-Antichymotrypsins*

(Received for publicat ion, June 22,1989)

Harvey RubinS, Zhi mei Wang& Ell iot t B. Nickbargg, Sean McLarneyS , Nir injini NaidooQ,

Oeyvind L. Schoenbergerg, Jeffrey L. JohnsonQll, and Barry S. Cooperman

From the $Department

of

Medicine, University

of

Pennsylvania, Philadelphia,

Pennsylvania

19104-6073 and the SDepartment

of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323

Huma n al-ant ichymotrypsin has been cloned, se-

quenced and expressed in

Escherichia coli

and recom-

binant protein as well as point-specif ic mutan ts have

been purified and characterized. The corrected gene-

deduced amino acid sequence has 45% overal l ident ity

with al-protease inhibitor, which is higher than the

42% previously reported (Chandra, T., Stackhouse, R.,

Kidd, V. J. , Robson, J. H., and Woo , S. L. C. (1983)

Biochemistry 22, 5055-5060). Recombinan t ant ichy-

motrypsin (rACT) is similar to natural ant ichymotryp-

sin with respect to the specif ic i ty of i ts interact ions

with proteases. I ts second-order rate constant for as-

sociat ion with bovine chymotrypsin is 6-8

X

10 M-

s-l, which is ident ical to that of the serum-derived

inhibitor. Site-specif ic mutagenesis has been used to

produce two variants of rACT in which the Pl posit ion

has been changed from leucine to either methionine

(L358M-rACT) or arginine (L358R-rACT). L358M-

rACT has a specif ic i ty of inhibitory act iv ity toward

serine proteases closely similar to that of nat ive rACT .

By contrast, the specif ic i ty of L358R-rACT is quite

dif ferent f rom that of nat ive rACT, mos t notably in

eff ic ient ly inhibit ing trypsin and human thrombin

while showing a decreased abil i ty to inhibit chymo-

trypsin.

cul-Ant ichymotrypsin (ACT) is a serine protease inhibitor

(serpin)

(1).

In i ts nat ive, c irculating form, i t is a glycoprotein

of between 55,000 and 66,000 daltons, with the variat ion

attr ibuted to microheterogeneity in glycosylat ion (2). I t is

synthesized predominant ly in the l iver and has also been

reported in mas t cel ls, s inus hist iocytes, endothelial cel ls, and

in cel ls of the hist io/mono cyt ic l ine (3). In response to inf lam-

matory st imuli , plasma levels of ACT increase more than 4-

fold within several hours (3). A famil ial form of ACT def i-

c iency has been described in which heterozygotes have 50%

of normal circulating levels (4). No homozygo te has been

reported, and such a genotype may be incompatible with l i fe

(4).

The precise biological role of ACT has not been determined.

Based on its rapid rate of associat ion with ca thepsin G, i t

may regulate the act iv ity of this neutrophil serine protease

* This work was supported by a grant (to H. R.) from H & Q Life

Sciences (San Francisco, CA). The costs of aublicatio n of this article

were defrayed in part by the payment of page charges. This article

must therefore be hereby marked advertisement in accordance with

18 U.S.C. Section 1734 solely to indicate this fact.

11 Kodak Fellow.

The abbreviations used are: ACT, antichymotrypsin; PBS , phos-

phate-buffered saline; rACT, recombinant antichymotrypsin; SDS,

sodium dodecyl sulfate; FPLC, fast protein liquid chromatography.

(1). Howev er, other targets are also possible. Chymo trypsin-

l ike enzym es and their inhibitors have been ident ified in a

wide variety of normal and abnormal biological processes

including modulat ion of cel lular funct ions (5-9), DNA binding

(lo), inhibition of certain parasite function s (11-15) an d

processing of vasoconstrictor proteins (16). ACT appears to

be a componen t of the amyloid deposit in Alzheimers plaques

(17) and is present in various carcinomas (18,19) and in some

tissues of the reproduct ive system (20, 21).

ACT forms SDS-stable complexes with i ts target enzymes

(22,23), which is a general property of serpin/serine protease

interact ions. Lit t le of a detai led nature is known about the

nature of these complexe s. Although high resolut ion crystal

structures have been determined for a form of the related

serpin, human c Yl-proteas e inhibitor, in which the Pl-Pl

pept ide bond has been hydrolyzed (24), as well as for com-

plexes o f serine proteases and some smaller pept ide inhibitors

(25-29), no direct structural studies of ACT alone or as a

complex with a serine protease have been reported.

In this paper we report the cloning, expression and muta-

genesis of the human cul-ant ichymotrypsin gene, and the

purif icat ion and characterizat ion of both the recombinant

protein and of two variants of the recombinant protein pro-

duced by mutat ion at i ts Pl s ite.

MATERIALS AND METHODS

Isopropyl-@-thiogalactopyranoside, EcoRI, PstI, HindIII, calf in-

test inal alkal ine phosphatase,

T4 DNA

polymerase, mung bean nu-

clease, Klenow fragment, pUC19, and pKK233 were obtained from

Promega (Madison, WI). Diamino benzidi ne, DNA-cellulose, bovine

pancreatic chymotrypsin and trypsin, and all chromophoric protease

substrates were obtained from Sigm a. Human thrombin was from

Sigm a or Behring Diagnostics. Porcine pancreatic elastase was from

Behring Diagnostics. Human neutrophil elastase was from EPC (Pa-

cific, MO). DH5, JM101, and JM105 cells were obtained from the

Cell Center of the University of Pennsylvania. pINomp/Ncol/b, a

secret ion vector that al lows fusion of a cloned protein to the

omp

leader peptide, was obtaine d from Professor John Collins and Dr.

Gerhard Gross (Gesellschaft fiir Biotechnologische Forschung,

Braunschweig, Federal Republic of Germany). pKC30 and Esche-

richiu coli N4830-1 were from Pharmacia LKB Biotechnology Inc.

The pAR 3039 vector originates from Studier and Moffat (30) as

described.

Human serum ACT was prepared using a procedure based on the

work of Tsuda et al. (31). Briefly, this method affords pure ACT in

three steps , batchwise elut ion from DNA-cellulose, G-150 chromatog-

raphy, and NaCl gradient elution from DNA-cellulose. A full descrip-

tion of this procedure will appear e lsewhere.3

Plasm id constructions were carried out followi ng Maniatis et al.

(32).

The nu mbering of the reactive site sequence follows Schechter

and Berger (57).

3 L. Kilpatrick, J. L. Johnson, T. F. Clifford, B. S. Cooperman, S.

D. Douglas, and H. Rubin, manuscript in preparation.

1199

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3/10

1200 Recombinant Native and Variant Human cul-An tichymotrypsins

Identif ication and Sequencing of the Gene for Human ACT

A human liver cDNA library in the phage expression vector Xgtll

(generously provided by Mitchel l Weiss, Department of Human Ge-

netics, University of Pennsylvania) was screened according to the

method of Young and Davis (33) with polyclonal antisera raised

against Cl esterase inhibitor (DAKO, Santa Barbara, CA), a related

human serine protease in hibitor. Positives were picked, rescreened,

and plaque-pu rified. DNA sequencing was performed with the chain

termination method (34) using oligonuc leotide primers obtained from

the Nucleic Acid Synthesis Center of the Wistar Institute (Philade l-

phia, PA).

Expression Systems

The EcoRI insert from the recombinant Xgtll was subcloned into

pUC19. p KK23 3 was digested with HindIII, the overhanging ends

were filled in by treatment with Kleno w fragment in the presence of

the four deoxynucleotide triphosphates, and dephosphorylated with

alkaline phosphatase. The EcoRI-EcoRI fragment containing the

entire ACT-coding sequence was removed from the pUC19 vector,

isolated by agarose gel electrophoresis, treated with mung bean nu-

clease to generate a blunt end fragment in the correct reading frame,

and ligated to the modifie d pKK2 33 vector described above. The

recombinant was denoted pKKACT and yielded recombinant protein

denoted rACT-1. The construction is described in Fig. 1.

A heat-inducible, high expression vector denoted pT7PL was pre-

pared by first placing the Shine-Da lgarno sequence and start codon

from the T7 vector pAR30 39 upstream to the coding sequence of the

antichymotrypsin gene and then placing the gene with these heter-

ologous regulatory sequences under the control of the Pn promoter in

pKC30 . The recombinant was denoted pACT2 and yielded recombi-

nant protein denoted rACT-2. The construction is described in Fig.

2.

pKT280 (Clontech) was digested with PstI, phenol/chloroform-

extracted and ethanol-precipitated. The 3 overhanging ends were

removed with T4 DNA polymerase using 2 units of enzyme/pg DNA

in the presence of 3.3

m M

dNTPs. The vector was then treated with

calf alkaline phosphatase. The EcoRI-EcoRI fragment c ontaining the

entire antichymotrypsin-coding sequence was isolated by agarose gel

electrophoresis and the 5 overhanging ends filled in with Klenow

fragment as described above. The resulting DNA was blunt-end

ligated to the vector prepared as described, yielding the recombinant

labeled pKTACT.

Site-directed

Mutagenesis

Site-directed mutagenesis was carried out using the Bio-Rad Ml3

in vitro mutagenesis kit and the synthetic DNA primers (5-CTA-

ATGCAGACATGAGGGTGATT-3 for L358M and 5-TGCAGA-

ACGGAGGGT-3 for L358R). The altered genes were excised from

double-stranded Ml3 with EcoRI and inserted into pKK2 33 as de-

scribed for the wild-type construction, yielding recombinants denoted

pKKACT-M and pKKACT-R for the methionine and arginine mu-

tants, respectively. Both mutations were confirmed by DNA sequenc-

pKK 233-2

22

. .CC ATG GCT GCA GCC AAG CTT

full length gene

ECORI

EWRI

1yifi;K

I

CAP

CC ATG GCT GCA GCC AAG CT

AA l-K CTC TGC CAC CCT AAC . . .

I

f.kNlQ Bean Nucleaee

C CTC TGC CA C CCT AAC

Leu Cys His Pro Asn

I

T-

ATG GCT GCA GCC AA0 CTC CTC TGC CAC CCT AAC.. .

Met Ala Ala Ala Lys Leu Leu Cye His Pro Asn...

FIG. 1. Scheme for the construction of the expression plas-

mid in pKKACT.

Ea....

BamHl

Klenow

CAP

full length gene

EcoRr

T

EmRI

AAI-X , CTC. TGC, GAG....

+

Mung Bean Nuclease

TC. TGC. GAG....

I

1 ligation

i

Xbe I and EmRV

Hpa I

Xbal D EmRV

CAP

1

Klenow

Xbal s EcoRv

t ligation

FIG. 2. Scheme for the construction of the expression plas-

mid in PACTS.

ing. The corresponding proteins were denoted L358M-rACT-1 and

L358R-rACT-1, respectively.

Sma ll Scale Growth Conditions and Extraction

Fresh overnight cultures of JM105 transformed with pKKA CT,

pKKACT-M, or pKKACT-R were diluted to 1.5% in LB broth

containing ampi cillin (sodium salt, 0.1 mg/ml) and grown to an

ODW., of 0.3, induced with 1.25

m M

isopropyl-@thiogalactopyran-

oside and grown for an additio nal 5 h. The cells were pelleted and

then disrupted in a French press. The ACT proteins purified from

these transformed cells are denoted rACT-1, L358M-rACT-1, and

L358R-rACT-1, respectively.

Fresh overnight cultures of N4830-1 were transformed with

pACT2, grown overnight at 30 C, diluted to 1% in LB broth contain-

ing ampic illin (sodium salt, 0.1 mg/ml) and grown to an ODsw., of

0.2, then shifted to 42 C and grown for an additio nal 5 h at 42 C.

The cells were pellete d and disrupted as above. The ACT protein

purified from this transformed cell is denoted rACT-2.

Fresh overnight cultures of DH5 cells transformed with pKTACT

were diluted to 1.5% in LB broth containing ampic illin (sodium salt,

0.1 mg/ml), grown for 7 h, and harvested by centrifugation. The

washed cell pellet was suspended in 20% sucrose, 50

m M

Tris, pH

7.5, 10

m M

EDTA, shaken for 7 min at room temperature, and

centrifuged at 13,000 x g for 10 min. The pe llet was rapidly resus-

pended in double-d istilled water, and frozen, thaw ed, and sonicated

three times. The resulting mixture was centrifuged at 13,000 X g for

10 min, an d the supernatant was saved.

Western Blots

Comme rcial antisera (DAKO) were absorbed prior to use according

to the followin g method. An overnight culture of JMlOl (200 ml) was

pellete d for 5 min at 4 C, rinsed with phosphate-buffered saline,

resuspended in 6 ml of cold phosphate-buffered saline and then frozen

and thawed three times in Dry Ice-ethanol. The resulting mixture

was sonicated six times for 30 s each and pelle ted in a microcentrifuge

at room temperature for 5 min. The supernatant was then diluted to

2% in phosphate-buffered saline. 1.5 ml of 2% supernatant was added

to a piece of nitrocellulose paper cut in 2.5

x

2.5-cm squares. This

mixture was shaken at room tempe rature for 1 h. The nitrocellulose

was then rinsed twice in phosphate-buffered saline. The Escherichia

coli extract-saturated paper was added to 20 ml of rabbit anti-

antichymotrypsin serum, diluted 1:600 in Blotto (2% dry milk in

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Recombinant Native and Variant Human al -Antichymotrypsins 1201

FIG. 3. DNA and amino acid se-

quences. Comparison of gene-deduced

cul-antichymotrypsin

sequences

(our

data and those of Ref. 39) and of the al-

protease inhibito r sequence (41). Posi-

tions of base pair deletions and inser-

tions are indicated by arrows. Residues

that are identical between our corrected

al-antichymotrypsin sequence and the

al-protease inhibitor sequence are indi-

cated by asterisks.

This

J c

CTG TCT CTG GGG GCC CAT AAT AC C ACC CTG ACA GAG ATT CTC A AA GGC C;, AAG

work

Lsu Ser Leu Gly Ala His Asn Thr Thr Leu Thr Glu Ile Leu Lys Gly ~eu

L Y S

Chandra

CTG TCT CTG GGG GCC CAT AAT ACC ACC CTG ACA GAG ATT CTC AAG GCC TCG AGT

Leo Ser Leu Gly Ala His

Asn

Thr Thr Le u Thr Glu Ile Leu Lys Ala Ser Ser

al PI

CTC TCC CTG GGG ACC A AG GCT GAC ACT C AC GAT GAA ATC CTG GAG GGC CTG AAT

Leu Ser Leu Gly Thr Lys Ala Asp Thr His A sp Glu Ile Leu Glu Gly Leu Asn

This CAG AGC TTC CAG CAC CTC

work

Phe Asn

Leu Thr Glu Thr S er Glu Ala Glu Ile His Gln Ser P he Gln His Leu

Chandm TCA CCT CAC GGA GAC TTA CTG AGG CAG AAA TTC ACT CAG AGC TTC CAG CAC CTC

Ser Pro His Gly Asp Leu Le u Arg Gln Lys Phe Thr Gln Ser Phe Gln His L eu

al PI

TTC AAC CTC ACG GAG ATT CCG GAG GCT C AG ATC CAT GA A GGC TTC CAG GAA CTC

Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gin Glu Leu

This

CTG C:C A CC CTC AAT CAG TCC

work

Leu Arg Thr Le u Asn

Gln Ser

Chandra CGC GCA CCC TCA ATC AGT TCC

Arg Ala Pro Ser Ila Ser Ser

al PI

CTCGT ACC CTC AAC CAG CCA

Leu Arg Thr L eu Asn Gln Pro

TABLE I

Purification of recombinant antichymotrypsin from two E. coli

expression systems

step

Antichy- Total

b Yield

Purification

motrypsin protein

factor

w mg

%

E. coli pKKACT(2-45-32)

Crude lysate

0.47d

300 100

Fast Q 0.41 33 86 7.9

DNA-cellulose 0.20 0.30 42 430

E. coli pT7PL

Crude lysate NW 2500

Fast Q 132 880 100

DNA-cellulose

107 122 81 5.8

'The amount ofantichymotrypsin was determinedbytitration of

bovine chymotrypsin as described in the methods section.

*The amountofto talprotein wasdetermined usingthemethodo f

Bradford (43).

From 3.1 g of cell paste (wet weight).

The amount of antichymotrypsin in the crude lysate was esti-

mated using Mono Q chromatography followed by titration as de-

scribed under Materials and Methods.

e From 19.1 g of cell paste (wet weight).

Not determined.

1.0% Triton, 0.05 M Tris, 10.0 mM EDTA) and swirled for 1 h at

room temperature. Proteins were transferred to nitrocellulose paper

followin g the procedure of Tobin et al. (35). The resulting Western

blots were stained using the ABC Vectastain kit (Vector Laboratories)

and the color was developed with diaminob enzidine .

ACT and Antitrypsin Activity in Crude Lysates

ACT activity could not be directly measured in crude bacterial

lysates because of a large background inhibitory activity in the lysate

itself. The background activity was separated from the antichymo-

trypsin by anion-exchange chromatography using a Mono Q HR5/5

anion-exchange FPLC column (Pharmacia LKB Biotechnology Inc.)

fitted into a LKB 2150 pump, 2152 gradient controller, and Waters

440 UV absorbance detector with an extended wavelength module .

Chromatography was typically conducted on the extract from 200 mg

of cells. The separation involved an isocratic wash (5 min) with 50

mM Tris-Cl buffer, pH 7.5, containing 50 mM KCl, followe d by a

linear gradient of KC1 (50-350 mM in 30 min) at a flow rate of 1.0

ml/min . Protein absorbance was monitored at both 214 and 280 nm.

Fractions (1.0 ml) were collected and assayed for ACT or antitrypsin

activity, measured as the inhib ition of the chymotrypsin-catalyzed

hydrolysis of substrate N-succinyl-A-A-P-F-p-nitroanilide (36) or of

trypsin-catalyzed hydrolysis of substrate N-Bz-P-F-R-p-nitroanilide.

A typical chymotrypsin assay contained (in 1.0 ml) 100 mM Tris-Cl

buffer, pH 8.3, 0.005% (v/v) Triton X-100, bovine pancreatic chy-

motrypsin (18 pmol) an d column eluate (0.005-0.5 ml). The assay

mixture was preincubated at room temperature for 5 min, substrate

(0.01 ml of a 10 mM solution in 90% dime thyl sulfoxide) was added,

and remaining chymotrypsin activity was determined by the rate of

change in Ano ,,,,

caused by the release of p-nitroanilide. A typical

trypsin assay contained (in 1.0 ml) 100 mM Tris-Cl buffer, pH 8.3,

0.005% (v/v) Triton X-100, bovine trypsin (8.6 pmol) and sample

(0.005-0.5 ml). The assay mixture was preincubated at room temper-

ature for 10 min, substrate (0.02 ml of a 15 mM solution in 90%

dimethyl sulfoxide) was added, and remainin g trypsin activity was

determined as above. Measurements of optical absorbance were con-

ducted at 25 C using a Hewlett-Packard 8452A spectrophotometer

fitted with a temperature controlled sample compartment.

The amount of active rACT-1, rACT-2, or L358M-rACT-1 present

was determined by titration of a solution of chymotrypsin of known

concentration and activity with varying amounts of partially purified

rACT fractions. The amount of active chymotrypsin present after

incubation with the inhibitor-containin g solutions was then deter-

mined using the chymotrypsin activity assay. The amount of active

L358R-rACT-1 present was determined in a similar manner by titra-

tion of a solution of trypsin of known concentration, using the trypsin

activity assay. Concentrations of chymotrypsin and trypsin were

determined using the active-site titration method of Ardelt and Las-

kowski (37).

Purification and Characterization of Recombina nt Antichymotrypsins

Large-scale Growth of E. coli-E. coli JM105 strains were grown to

a density of 4-5 ODhho., in LB mediu m containing ampic illin (sodium

salt, 0.1 mg/ml) and glucose (0.1% w/v) at 37 C in a 15-liter carboy

fitted with an oxygen bubbler. Cells were harvested by passage

through a Sharpless continuous-flow centrifuge. 3.5-5 g of cell paste

(wet weight) were obtained/ liter of culture. E. coli N4830-1 trans-

formed with pACT2 was grown in LB medium containing ampicil l in

(sodium salt, 0.1 mg/ml) in a 15-liter carboy fitted with a heating

coil, sampling tube, temperature probe, and an air bubbler. The

mediu m was inoculated with an overnight culture (200 ml) that had

beengrownat aconstanttemperatureof30 "C.Growthwascontinued

at 30 C for 1 h until the culture had reached an ODsoo.,,, of 0.17. The

temperature of the mediu m was shifted to 42 C by pumpin g steam

through the heating coil for a period of two min and then maintained

at that temperature by a heating circulation bath for 6 h until the

culture had reached an OD,,, of 0.9. The cells were harvested with

a Sharpless centrifuge as described above. 1.2 g of cell paste (wet

weight) were obtained/lite r of culture.

Extraction and Column Chromatographies-Purifications of rACT-

1, rACT-2, L358M-rACT-1, and L-358R-rACT-1 were all carried out

in an essentially identical manner, with the exception that in the

latter case an antitrypsin rather than an antichymotrypsin assay was

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1202

Recombinant Native and Variant Human (~1 -Antichymotrypsins

- -69

-

:: -46

- -30

ABCD

FIG. 4. SDS-stacking gel electrophoresis of purified recom-

binant antichymotrypsin. Lane A, 5 pg of purified antichymotryp-

sin from fractions 49-50 of the DNA-cellulose column; lane B, 25 Kg

of combine d fractions 50-58 from the Fast Q column; lane C, 50 Kg

of crude lysate;

lane D,

molecular weight markers. Protein was visu-

alized with a Coomassie Blue stain.

used to detect and quantitate recombinant inhibitor. All purification

steps were carried out at 4 C. In a typical preparation of rACT-1,

cell paste (3.1 g) was dispersed in 10 mM potassium phosphate buffer,

pH 6.9 (25 ml) and lysed by three passes through a French press at

10,000 psi and 4 C. Cell debris was removed by centrifugation at

30,000 x g for 30 min a t 4 C. The supernatant (25 ml) was loade d

onto a column (4.9 cm* x 37 cm) of Sepharose Fast Q (Pharmacia

LKB Biotechnolo gy Inc.) that had been equilibra ted to 50 mM Tris-

Cl, pH 7.5, containing 50 mM KCI. Protein eluted with a l inear

gradient of KC1 in 50 mM Tris-Cl, pH 7.5 (50-500 mM in 2 liters).

Fractions (15 ml) were monitore d for protein by AZBOnmnd assayed

for antichymotrypsin activity as described above. rACT-1 eluted at

approximately 200 mM KCl. Fractions 50-58, containing rACT-1,

were combined and dialyzed against two volumes (2.5 liters each) of

10 mM potassium phosphate buffer, pH 6.9, over 48 h. The dialyzed

solution was then applied to a DNA-cellulose column (1.7 cm2 X 20

cm) that had been pre-equilibrated with 10 mM potassium phosphate,

pH 6.9, containing 10 mM KCl. After loading, the column was first

washed with the same buffer (20 ml). The column was eluted with a

linear gradient of KC1 (lo-400 mM, 300 ml) in the same buffer.

Fractions (8 ml) were assayed for protein and antichymotrypsin

activity as above. rACT-1 eluted between 350 and 400 mM KC1

(fractions 49-53). Fractions containing antichymotrypsin activity

were analyzed for purity by SDS-P AGE, performed according to

Laem mli (38). Fractions in the early portion of the antichymotrypsin

peak showed a higher level of purity than those in the later portion .

Each portion was concentrated by ultrafiltration using Amicon YM-

10 membranes and dialyzed overnight against 50 mM Tris-Cl, pH 7.5

(500 ml). In some cases recombinant proteins were further purified

on a FPLC MonoQ anion-exchange column, using the conditions

described above.

Autom ated Edm an sequence analysis on rACT-2, using the Milli-

gen/Biosearch 6600 Series Prosequencer of the Protein Facility of

the Dental School of the University of Pennsylvania, yielded a

sequence in full accord with that predicted in Fig. 2, begin ning with

the tripeptide Ala-Ser-Met.

Kinetics of Complex Formation

The rates of inhib ition by human serum ACT, rACT-1, rACT-2,

L358M-rACT-1, and L358R-rACT-1 of bovine chymotrypsin, bovine

trypsin, human thrombin, porcine pancreatic elastase, and human

neutroph il elastase w ere investigated at 25 C under second-order

conditions in reaction mixtures containing equim olar concentrations

of enzyme and inhibitor, or under pseudo-first order conditions with

an excess of inhibitor. As described above, enzyme and inhibitor were

incubated in 100 mM Tris-Cl buffer, pH 8.3, containing 0.005% (v/v)

Triton X-100 for varying periods of time, substrate was then added,

and the amoun t of remain ing active enzyme was determine d. Alter-

natively, at timed intervals aliquots of the inhibitor plus enzyme

solution were diluted into an assay solution containing the appropri-

ate substrate and protease activity was determined .

RESULTS

Cloning and Sequencing of the Gene for Antichym otrypsin-

The DNA sequence and the derived amino acid sequence of

the insert from one of the positive Xgtll cDNA clones con-

tained the entire coding region of mature human cul-antichy-

motrypsin . The inser t also included an extension on the 5-

end encoding additional amino acids that appear in the pre-

curso r of the mature protein. The mature, serum-derived

protein has been reported to contain 398 amino ac ids (MI

45,031), starting from the tripeptide Asn-Ser-Pro (Fig. 2) at

the amino terminus (39). More recently, we3 have demon-

strated the presence of a second form of mature protein that

includes two additional amino acids, His-Pro, at the NH,

terminus (Fig. 2). The reactive center, Pl-P l, Leu-Ser, is

found at positions 358-359. The COOH-terminal sequence is

in agreement with Hill et al. (40) and the remainder of the

sequence is in agreement with Chandra

et al. (39)

except for

the 15 amino acids from position 77 to 91 and the six amino

acids from 98 to 103. These differences can be explained by

three inse rtions and three deletions of single bases within the

Chandra sequence. The sequence reported here displays a

high degree of similarity with al-protease inhibitor in this

region (Fig. 3) and raises the overall identity with al-protease

inhibitor by 17 residues, to 44.5 . The intron/exon structure

of these proteins has recently been reported (42) to be iden-

tical, with five exons separated by four introns. Comparison

of the antichymotrypsin and al-protease inhibitor sequences

exon by exon shows an interesting pattern. Exons II, IV, and

V have large percentages of identica l amino acid residues: 51,

44, and 46 , respectively. However, exon III has only 33

identity. The reactive center of the inhibitor is found in exon

IV. Fina lly, we note that, in comparison with the earlier work

of Chandra

et

al. (39), our sequence shows a proline at position

44 rather than a leucine, a leucine at 174 rather than a proline,

an alanine for valine at 336, and a leucine for serine at 338.

The latter two amino acid substitutions are also reported by

Hill

et al. (40).

Expression of the ACT Gene-Four different E. coli vectors,

two secretion systems (pINomp/iVcoI/b and pKT280) and

two non-secretion systems (pKK233 and pT7PL), were eval-

uated for expression of recombinant human antichymotryp-

sin. More than 100 recombinants in a modified pINomp vector

were screened and yielded plasmids with the insert in the

wrong orientation in every instance (results not shown). We

presume that the correct orientation constructs lead to high

level production of a toxic gene product. A second secretion

plasmid, pKT280, in which expression is driven from the /3-

lactamase promoter and which encodes part of the signal

sequence of p-lactamase, was also examined. The construct

yielded full length ACT in low yield with an amino-terminal

extension coding for an additional eight amino acids, His -

Pro-Gln-Phe-Leu-Cys-His-Pro , the first four originating from

the vector and the following four from the precursor of anti-

chymotrypsin.

The expression plasmid pKK233, utilizes the strong trp-lac

promotor and yielded a recombinant protein, migrating with

an apparent

M ,

of 45,000, that could be detected on Western

blots of crude extracts, using affinity purified antibody raised

against al-antichymotrypsin. The construction of the ex-

pressed protein contains an amino-term inal extension of 10

residues (Fig. 1).

The highest level of expression was obtained with pACT2,

which utilizes the PL system (Fig. 2).

Purif icat ion of Recombinant Antichymotrypsins-The

pu-

rifications of rACT-1 and rACT-2 are summarized in Table

I, from which it is clear that much higher levels of expression

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B

Recombinant Native and Variant Human cul Antichymotrypsins

r ACTI L358R-r ACT I

L358R-rACT I

lhrombin

thrombln

tryps1n

-200 K

-El\ 1;; ;

I;;y(

-69 K

----

i *

-46 K

-

--

ibIg/ -46K

b

-30 K

-30 K

ABCDEF ABCDEF ABCDEFG

L358M-rACT I

chymoirypsin

trypsin

,-200

K

-92 K

-69 K

%IL -_

-45 K

-30 K

ABCD E F ABCDE F

rACT- I

L3 38M-rACT I

porcme poncreotlc elostose

porcine poncreotlc elostose

I gKK

-200

K

ye -69K -

-92 K

-69 K

&u- -46K

-k.raDD-45 K

--30K

-30 K

ABCDEF

ABCDEFG

FIG. 5. Western blots of crude recombinant antichymotrypsins in the presence or absence of various

serine proteases. Upper panel, rACT-1 interaction with thrombin: lane A, rACT-1 alone; lanes B-F, rACT-1

incubated with 0.1, 0.5, 1, 5, and 10 ~1 of thrombin, respectively; L358R-rACT-1 interaction with thrombin: lane

A, L358R-rACT-1 alone; lanes B-F, L358R-rACT-1 incubated with 0.1,0.5,1,5, and 10 ~1 of thrombin, respectively;

L358R-rACT-1 interaction with trypsin: lane A, L358R-rACT-1 alone; lanes B-G, L358R-rACT-1 incubated with

0.1, 0.5, 1, 2, 5, and 10 ,ul of trypsin, respectively. Center pan el, L358M- rACT-1 interact ion with chymotrypsin:

lane A, L358M-rACT-1 alone; lanes B-F, L358M-rACT-1 incubated with 0.1, 0.5, 1, 5, and 10 ~1 of chymotrypsin,

respectively; L358M-rACT-1 interaction with trypsin: lane A, L358M-rACT-1 alone; lanes B-F, L358M-rACT-1

incubated with 0.1, 0.5, 1, 5, and 10 ~1 of trypsin, respectively. Lower panel, rACT-1 interaction with porcine

pancreatic elastase: lane A, rACT-1 alone; lanes B-F, rACT-1 incubated with 0.1, 0.5, 1, 5, and 10 ~1 of porcine

pancreatic elastase, respectively. L358M-rACT -1 interacti on with porcine pancreatic elastase: lane A, L358M-

rACT-1 alone; lanes B-G, L358M-rACT-1 incubated with 0.1, 0.5, 1, 2, 5, and 10 ~1 of porcine pancreatic elastase,

respectively. The foll owin g stock protease solutions were used in these experiments: thromb in, 0.12 mg/m l; trypsin,

1 mg/m l; chymotrypsin, 1 mg/m l; porcine pancreatic elastase, 1 mg/m l. Crude antichymotrypsins were prepared

from extracts of 0.5-1.0 ml of bacterial culture.

are achieved with the second expression system . In both cases,

SDS-PAG E analysis of protein gradient-eluted from the

DNA-cellulose column (see Materials and Methods) showed

a single band at approxim ately 45,000 daltons (Fig. 4). Puri-

fication results sim ilar to those for rACT -1 were obtained for

L358M-rACT-1 and L358R-rACT-1.

Formation

of

SDS-Stable Complexes between Recombinant

Antichymotrypsins and Serine Proteases-The complex that

human serum ACT forms with chymotrypsin is stable in SDS-

polyacrylam ide gels and migrates at a higher molecular weight

than ACT itself (23). We used Western blots of Laemmli gels

(Fig. 5) to determine whether or not SDS-stable complexes

are formed between rACT (and rACT variants) and proteases,

giving the results summarized in Table I I4 As may be seen,

the complexes themselves appear to be substrates for the

In addition to the results presented in Table II, SDS-stable

complexes were shown to be formed betwee n rACT-1 and cathepsin

G and between L358R-rACT-1 and kallikrein, but not between

L358M-rACT-1 and Clr.

N. Schechter, personal communication.

corresponding uncomp lexed protease s, since they are readily

degraded as the [protease]/[inhibitor] ratio is increased.

The formation of serine protease-rACT complexes was

examined using either crude extracts containing rACTs or

highly purified rACTs . In the former case, such experiments

provided a useful screen for the expression of active protein.

As the abili ty of a rACT mutant to form an SDS-stable

complex with a protease correlates very well with its affording

a measurable rate of protease inhibition (Table II), we con-

clude that the mutant rACTs inhibit proteases by the same

mechanism as does human serum ACT . The one apparent

exception to the above noted correlation is the interaction

between L358M-rACT-1 and porcine pancreatic elastase.

Here we see evidence for complex formation (Fig. 5) but were

unable to meas ure a rate of protease inhibition. How ever, as

we would not detect inhibit ion from a rate constant that was

more than lo-fold less than that for rACT -1, this exception

ma y be more apparent than real.

It is interesting to note that when identical Wes tern blots

were reacted with the polyclonal serum raised against Cl

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Recombinant Native and Variant Human al -Antichymotrypsins

TABLE I I

Interact ion of recombinant ant ichymotrypsins and serine proteases

Formation of SDS-stable complexes and inhibit ion rate constants (measured at 25 C, uH 8.3). +. SDS-stable

complex formed, -, SDS-stable complex not formed, N D, not determined.

.

rACT-1 or rACT-2 L358M rACT L358R rACT

Enzyme Substrate (f inal molarity)

Molarity

k,

Molarit

k,

Molarity

k

IZM nhf I IM

Bovine chym otryps in Sue-A-A-P -F-p-nitroanilide 9 6-8 x lo5 (+) 18 3 X lo5 (+) 72 1 x 10 (+)

(0.2

mM) 18 360

[2.2 x lO]C

Trypsin

N-p-Tos-G-P-R-p-nitroanilide

172


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Recombinant Native and Variant Human Lul-Antichymotrypsins

1205

0

0

(4

80

0

(W

100

200 300

Time (min)

0

fa)

0 50 100 150 200

Time (min)

[l]/[E], (Molar Ratio) [l]/[E], (Molar Ratio)

FIG. 7. Stoichiometry of inhibit ion of chymotrypsin activity by L358R-ACT-1 and of porcine

pancreatic elastase activity by rACT-2. A: upper panel, chymotrypsin inhibit ion by L358R-ACT-l. The

concentration of chymotrypsin was held constant at 360

nM. [I]/[E]

ra io values were 0 (a); 0.5 (b); 1.0 (c); 2 .0

(d). Lower panel, plot of final activities uersus [I]/[E] ratio. B: upper panel, porcine pancreatic elastase inhibit ion

by rACT-2. The concentration of elastase was held constant at 39

nM.

[I]/[,??] ratio values were 0 (a); 1.0 (b); 2.0

(c); 3.0 (d); 4.0 (e); 5.0 (f). Lower panel, plot of final activities uersus [I]/[E] ratio. Very similar results were

obtained when human serum ACT replaced rACT-2.

0

(WW

o- 150 200

time (min)

FIG. 8. Second order kinetics of inhibit ion of chymotrypsin

by L358R-ACT-l. Reaction mixtures contained equimola r initia l

concentrations of enzyme and inhibitor. Curve a, [E] = [I] = 72 nM;

curue b, [E] = [I] = 360 nM. Curve c is a control measuring the

activity of chymotrypsin (360

nM)

in the absence of inhibitor.

More direct evidence for such a model in the case of porcine

pancreat ic elastase and human serum ACT comes from the

observat ion of Mori i and Travis (23) that elastase cleaves the

inhibitor between the P5 and P4 posit ions, thereby inact ivat-

ing it.

k,

h

- um

E+I k_ E.1 A (E.I),/

1

\

-E+Ii

k,

S C H E ME 1 .

Serpin as a suicide inhibitor. E is protease, I is serpin,

(EI)b is the irreversibly formed inactive complex. It is the inactive

inhibitor.

As has been pointed out by Fish and Bjork (47), when the

model applies the serpin may be formally considered as a

suicide inhibitor. An analytical solution for the classica l

scheme of suicide inhibition shown below (Scheme 1) has

been presented by Waley (48). Applying this solut ion to the

results in Fig. 8 permits evaluat ion of an apparent second

order rate constant for react ion of L358R-ACT-1 and chy-

motrypsin, equal to kJ~&/[(k-~ +

kz)(k3 + k4)],

of 1

X

lo4 M-

s-l. Since full inhibition is obtained at a 1:l ratio of serpin to

protease when 12s


9/10

1206 Recombinant Native and Variant Human cul Antichymotrypsins

matic activity on incubation of porcine pancreatic elastase

with rAC T-2 is 8.0 x lo3 M- s- which, corrected for (E.I),

partitioning by multiplying by the term (k3 + 124)/124Scheme

l), y ields an apparent second-order rate constant for @.I),

formation of 2.0 X lo4 M-' s-i. We obtained similar results

when human serum ACT was used in place of rACT-2. Laine

et al. (49) have reported that an I:E ratio of 5.5 was required

for full, irreversib le inhibition by human serum ACT of por-

cine pancreatic elastase. However, in this earlier work, per-

formed under conditions similar to our own, only a 60-min

incubation was used before inhibition was measured. As seen

in Fig. 7B, such an incubation time would underestimate

inhibition obtained at lower concentration of inhibitor and

lead to an overestimate of the amount of inhibitor required

for full inhibition of elastase activity.

DISCUSSION

A major focus of current studies of serpins and their target

proteases is the determination of the nature and extent of

contact regions between them, as a means of understanding

the specificities of such interactions. The results described in

this paper make two important contributions to such under-

standing. They show first, that glycosylation of the serum

protein (which accounts for approximate ly 30 of its total

molecular weight) is not essential for the interactions of ACT

with serine proteases and second, that the importance of the

amino acid residue in the Pl position of a rACT in determin-

ing its ability to inhibit serine proteases varies with the serine

protease under consideration.

The important structural difference between human serum

ACT and either rACT-1 or rACT-2 is that the serum protein

is glycosylated whereas the rACTs are not. Despite this dif-

ference, the two kinds of ACT show similar specificities

toward both formation of SDS-stable complexes with serine

proteins and inhibition of protease activ ity (Table II). In a

closely related study (50), recombinant cYl-proteinase inhibi-

tor produced in E. coli was found to have an association rate

constant for neutrophil elastase (7.2 X lo6 M-l 6-l) only

slightly below that found for the human serum protein (1.1 X

lo7 M- s-l) . It may therefore be general that the pattern and

extent of glycosy lation is not a dominant factor in serpin

interactions with their target proteases.

Much of the literature on serpins has focused on the con-

tribution of the Pl side chain to the rate of formation and

stabi lity of serpin:ser ine protease complexes. The results ob-

tained in the present work, as well as the related work of

others, provide evidence that th is focus is more justified for

some serpin:ser ine protease interactions than for others.

Thus, the properties of L358R-rACT-1 support the conclusion

that an arginine in the Pl position is a major determinant of

serpin specific ity in both a positive and negative sense. This

single amino acid change turns rACT into a serpin capable of

inhibiting thrombin and trypsin but having a considerably

reduced (30-fold) rate of reaction with chymotrypsin. In fact,

even though rACT and antithrombin III have only limited

(28 ) overall identity (51), the specificity of L358R-rACT-1

resembles that of antithrombin III (an inhibitor having an

arginine in its Pl position), and the rate constants for inhi-

bition of thrombin by these two inhibitors are virtually iden-

tical, 4.3 x lo3 M- s-l for L358R-rACT-1 in the present work

and 2.5 x lo3 M- s-l for antithrombin III measured by others

under similar conditions (52). Similarly, human cyl-protease

inhibitor was shown to be converted from an antielastase to

an antithrombin by the naturally occurr ing Pittsburgh

M358R mutation, a mutation that also resulted in a 25-fold

reduction in the rate of inhibition of the chymotrypsin-like

enzyme, cathepsin G, and an 8000-fold reduction in the rate

of inhibition of human neutrophil elastase (53).

By contrast, even though human al-protease inhibitor is a

potent elastase inhibitor, has a methionine in its Pl position

and, as we have demonstrated above, a higher (44.5 ) overa ll

identity with rACT than antithrombin, the L358M mutation

of rACT-I fails to convert rACT into an effective inhibitor of

elastase. In related work, mutation of methionine 358 in

human cYl-protease nhibitor to a variety of other hydrophobic

residues, including valine, alanine, leuc ine, and isoleucine, has

only modest effects on the second order rate constant for

inhibition of human neutrophil elastase (50). Results obtained

using small synthetic oligopeptide substrates and inhibitors

(29) also suggest that the S l site of human neutrophil elastase

can accommodate a variety of hydrophobic amino acid side

chains. Therefore, antielastase activity, unlike antithrombin

activity, has no comparable dependence on the presence of a

particular residue in the P l position.

These latter results suggest that, for at least some serpin-

serine protease complexes, specificity-determining interac-

tions take place with serpin residues other than that in the

Pl position. A major question is the extent to which such

residues fall within the reactive site loop of protease inhibitors

(from position P6 to position P3 (29)) or in other portions

of the protein. X-ray structure determination of serine pro-

tease complexes with smaller protease inhibitors such as

bovine pancreatic trypsin inhibitor (25), turkey ovomucoid

third domain inhibitor (28), eglin c (26), and secretory leu-

kocyte protease inhibitor (27) have shown many contacts

between proteases and positions within the reactive site loop

of the inhibitor, and results with site-specific mutants (52,54,

55) and with small synthetic oligopeptide substrates and

inhibitors (29, 58, 59) provide evidence that such contacts

make important contribut ions to binding. On the other hand,

some of the x-ray results (29) also show evidence for a second

contact domain on inhibitors well outside the reactive site

loop. Furthermore, Mierzwa and Chan (56) have presented

evidence based on chemical modification experiments that

elastase interacts w ith al-protease inhibitor at residues that

are far from the Pl site. We are presently undertaking a series

of experiments combining studies of the properties of point-

specific mutants with chemical modification and x-ray crys-

tallographic analysis to close ly define the structural details of

antichymotrypsin-chymotrypsin interaction. This work forms

part of our overa ll goals to investigate the interaction of

antichymotrypsin with its natural targets, study the regula-

tion of the gene in response to mediators of inflammation,

and evaluate the biological role and potential therapeutic

applications of antichymotrypsin.

REFERENCES

1. Travis, J., and Salvesen,G. S. (1983)Annu. Reo. Biochem. 52,

655-709

2. Tokes,Z. A., Gendler,S.J., and Dermer, G. B. (1981) J. Supramo l.

Strut. Cell. Biochem. 17,69-77

3. Berninger,R. W. (1985) J. Med. 16, 101-127

4. Erikson, S.,Lindmark, B., and Lilja, H. (1986) cta Med. Scandia

220.447-453

5. King, G. H., Gora linik, C. H., Kleinhenz, P. J., Marino, J. A.,

Sedor, J. R.. and Mahmoud, A. A. (1987) J. Clin. Znuest. 79,

1091-iO98

6. Braun, N. J., and Schnebli, H. P. (1987) Biol. Che m. Hoppe-

Seyler 368, 155-161

7. Camussi,G., Tetta, C., Bussolino,F., and Baglioni, C. (1988) J.

l&p. Med. 168, 1293-1307

8. Yavelow, J., Collins, M., Birk, Y., Troll, W., and Kennedy, A. R.

(1985)Proc. N&l. Acad. Sci. U. S. A. 82, 5395-53 99

9. Yavelow, J., Caggana, M., and Beck, K. A.

(1987) Cancer Res.

47, 1598-1601

atTENNESSEETE


http://www.jbc.org/

Downloaded

from


10/10

Recombinant Native and Variant Human al -Antichymotrypsins

1207

10. Tsuda, M., Umezawa, Y., Masuyama, M., Yamagu chi, K., and 34. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. N&l.

Katsunuma, T. (1987) Biochem . Biophys. Res. Commu n. 144, Ad. Sci. U. S. A. 74,5463-5467

409-414 35. Tobin, H., Staeh elin, T., and Gordon. J. (1979) Proc. Natl. Ad.

11. Braun-Breton, C., Rosenberry, T. L., and Pereira da Silva, L. Sci. U. S. A. 76, 4350-4354

(1988) Nature 332, 457-459 36. DelMar, E. G., Largman, C., Brodrick, J. W., and Geokas, M. C.

12. Banyal, H. S., Misra, G. C., Gupta, C. M., and Dutta, G. P. (1981) (1979) And. Biochem. 99, 316-320

J. Purasitol. 67, 623-627 37. Ardelt, W., and Laskowski, M., Jr. (1985) Biochemistry 24,5313-

13. Dluzewski, A. R., Rangachari, K., Wilson, R. J., and Gratzer, W.

5320

B. (1986) Exn. Parusitol. 62, 416-422 38. Laem mli. I-J. K. (1970) Nature 227.680-685

14. McKerrow, J.-H., Pino-Heiss; S., Lindquist, R., and Werb, Z.

(1985) J. Biol. Chem. 2 60, 3703-3707

15. Asahi, M., Lindquist, R., Fukuyama, K., Apodaca, G., Epstein,

W. L., and McKerrow, J. H. (1985) Biochem. J. 232, 139-144

16. Yanagisawa , M., Kurihara, H., Kimura, S., Tomobe, Y., Koba-

yashi, M ., Mitsui, Y., Yazaki, Y., Gota, K., and Masaki, T.

(1988) Nature 332, 411-415

17. Abraham, C. R., Selkoe, D. J., and Potter, H. (1988) Cell 52,

487-501

18. Tahara, E., Ito, H., Taniyama, K., Yokozaki, H., and Hata, J.

(1984) Human Puthol. 15,957-964

19. Ordone, N. G., and Manning, J. T. (1984) Am. J. Gastro. 79,

959-963

20. Schill, W. B. (1976) Andr ologia 8, 359-364

21. Casslen, B., and Ohlsson, K. (1981) Contraception 23, 425-434

22. Travis, J., Bowen, J., and Baugh, R. (1978) Biochemistry

17,

5651-5656

23.

Morii, M., and Travis, J. (1983) J. Biol. Chem. 258,12749-12 752

24. Lobermann, H., Tokuoka, R., Deisenhofer, J., and Huber, R.

(1984) J. Mol.. Biol . 177; 531-556

25. Marauart. M.. Walter. J.. Deisenhoffer. J.. Bode. W.. and Huber.

R. (1983) Acta Cry&ilogr. Sect. B. ktkct. C&&logr . Cryst:

Chem. 39,480-490

26. Bode. W.. Panamokos. E., and Musil. D. (1987) Eur. J. Biochem.

166,673-692

27. Fujinaga, M., Sielecki, A. R., Read, R. J., Ardelt, W., Laskowski,

M., Jr.. and James, M. N. (1987) J. Mol. Biol. 195,397-418

28. Grute, M. G., Fendrich, G., Huber, R., and Bode, W. (i988) Embo

J. 7,345-351

29. Bode, W., Meyer, E., Jr., and Powers, J. C. (1989) Biochemistry

28,1951-1963

30. Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Bio l.

189,

113-

130

31. Tsuda, M., Ohkubo, T., Kamigu chi, H., Suzuki, K., Nakasaki, H.,

Mitom i, T., and Katsunuma , T. (1982) J. Exp. Clin. Med. 7,

201-211

32. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular

Cloning : A Laboratory Manu al, Cold Spring Harbor Laboratory

Press, Cold Spring Harbor, NY

33. Young, R. A., and Davis, R. W. (1983) Proc. N atl. Acad. Sci. U.

S. A. 80, 1194-1198

39. Chandra, T., Stackhouse, R., Kidd,.V . J., Robson, K. J. H., and

Woo, S. L. C. (1983) Biochemistry 22,5055-5061

40. Hil l , R. E., Shaw, P. H., Boyd, P. A., Baumann, H., and Hastie,

N. D. (1984) Nature

311,175-177

41. Rosenberg, S., Barr, P. J., Najarian, R. C., and Hallew ell, R. A.

(1984) Nature

312, 77-80

42. Bao, J., Sifers, R. N., Kidd, V. J., Ledley, F. D., and Woo, S. L.

C. (1987) Biochemistry 26, 7755-7759

43. Bradford, M. M. (1976) Anal. Biochem. 72,248-254

44. Beatty, K., Bieth, J., and Travis, J. (1980) J. Biol. Ch em. 255,

3931-3934

45. Bjork, I., Jackson, C. M., Jornvall, H., Lavine, K. K., Nordling,

K., and Salsgiver, W. J. (1982) J. Biol. Chem . 257, 2406-2411

46. Bjork, I., and Fish, W. W. (1982) J. Biol. C hem. 257,9487-94 93

47. Fish, W. W., and Bjork, I. (1979) Eur. J. Biochem. 101, 31-38

48. Walev. S. G. (1985) Biochem. J. 227, 843-849

49. Lain;, A., Davril, M., Rabaud, M., -Vercaigne-Marko, D., and

Hayem, A. (1985) E ur. J. Biochem .

151,327-331

50. Jallat, S., Carvallo, D., Tessier, L. H., Roecklin, D., Roitsch, C.,

Dgushi, F., Crystal, R. G., and Courtney, M. (1986) Protein

Eng.

1,29-35

51. Bock, S . C., Skriver, K., Nielsen, E., Thegersen, H-C., Wiman,

B., Donaldson, V. H., Eddy, R. L., Marrinan, J., Radziejewska,

E., Huber, R., Shows, T. B., and Magnusson, S. (1986) Bio-

chemistry 25,4292-4301

52. Holmes, W. E., Lijnen, H. R., and Collen, D. (1987) Biochemistry

26,5133-5140

53. Travis, J., Matheson, N. R., George, P. M., and Carrell, R. W.

(1986) Biol. Chem. Hoppe-Seyler 367,853-859

54. Stephens, A. W., Siddi qui, A., and Hirs, C. H. W. (1988) J. Biol.

Chem. 263,15849-15852

55. Matheson, N.. Bathurst, I., and Travis. J. (1989) Biochem. Bio-

phys. R&. Cimmun. 154, 271-277

56. Mierzwa. S.. and Chan. S. K. (1987) Biochem . J. 246. 37-42

57. Schechter, I., and Berg&, A. (i967)Biochem . Biophys: Res. Com-

mun. 27, 157-162

58. Glover, G. I., Schasteen, C. S., Liu, W.-S., and Levine, R. P.

(1988) Mol. Zmmun ol. 25, 1261-1267

59. Schasteen, C. S., McLafferty, S. A., Clover, G. I., Han, C. Y.,

Mayden, J. C., Liu, W.-S., and Levine, R. P. (1988) Mol.

Zmmurwl. 25,1269-1275

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