Unit III Lecture 3

B. Tech. (Biotechnology) III Year V th Semester

EBT-501, Genetic Engineering

Unit III• Gene library: Construction cDNA library and genomic

library, Screening of gene libraries – screening by DNA hybridization, immunological assay and protein activity

• Marker genes: Selectable markers and Screenable markers, nonantibiotic markers,

• Gene expression in prokaryotes: Tissue specific promoter, wound inducible promoters, Strong and regulatable promoters; Increasing protein production;

• Fusion proteins; Translation expression vectors; DNA integration into bacterial genome; Increasing secretions; Metabolic load,

• Recombinant protein production in yeast: Saccharomyces cerevisiae expression systems

• Mammalian cell expression vectors: Selectable markers;

One of the aim of rDNA technology

Synthesis of large quantities of protein, either tostudy its properties or

because it has commercial value

In such instances, detectable synthesis is not sufficient: rather, it must be maximized.

Factors affecting the expression ofcloned genes.

Promoter strengthTranscriptional terminationPlasmid copy numberPlasmid stabilityHost-cell physiologyTranslational initiation sequences Codon choicemRNA structure

Maximizing protein synthesis by use of phage promoters in E.coli

There are three reasons for using a phage promoter.

First, such promoters are very strong, enabling large amounts of RNA to be made in vitro.

Secondly, the phage promoter is not recognized by the E. coli RNA polymerase and so no transcription will occur inside the cell. This minimizes any selection of variant inserts.

Thirdly, the RNA polymerases encoded by phages such as SP6, T7 and T3 are much simpler molecules to handle than the E. coli enzyme, since the active enzyme is a single polypeptide.

Control of expression of chloramphenicol acetyltransferase (CAT) in E. coli by three different promoters.

The levels of CAT are expressed as μg/mg total protein.

Vectors with strong, controllable promotersare used to maximize synthesis of cloned

gene products

• much of the interest in the application of recombinant DNA technology lies in the possibility of facile synthesis of large quantities of protein, either to study its properties or because it has commercial value.

• Many gene products can be toxic to the host cell even when synthesized in small amounts.

Strategy for regulating the expression of genes cloned into a pET vector

Tissue specific promoters

Several types of promoters regulate gene expression eg.Constitutive promoters

Tissue-specific or development-stage-specific promoters

Inducible promoters

Synthetic promoters

Tissue specific promotersAre promoter sequences on DNA of eukaryotic plant and

animal cells, which enable the expression of particular gene in the specific cell type

As cells of an organism contain same genetic information, some genes are turned on and others are turned off at different locations and times during the life cycle of an organism.

The transgenes driven by these type of promoters will only be expressed in tissues where the transgene product is desired, leaving the rest of the tissues in the plant unmodified by transgene expression

Some examples of tissue specific promoters in plants are

beta-amylase gene or barley hordein gene promoters (for seed gene expression)

tomato pz7 and pz130 gene promoters (for ovary gene expression)

tobacco RD2 gene promoter (for root gene expression)

banana TRX promotermelon actin promoter (for fruit gene expression)

Promoters used in animal cells

Promoters and enhancer sequences are used for driving transgene in different animal

Some examples of tissue specific promoters in animals arecytomegalovirus immediate-early gene

promoter (CMV)

human desmin (Des)

human alpha-myosin heavy chain (α-MHC)

rat myosin light chain 2 (MLC-2)

human cardiac troponin C (cTnC)

Gene transfer to animal cells

Why animal cells are used for cloning recombinant proteins

because they perform authentic post-translational modifications not carried out by bacterial cells and fungi

Objectives of gene transfer in animal cells

Study promoter function, reporter gene expression regulation

Expression of recombinant proteins in cultured cell line

Improve the quality of farm animals e.g. improvement of yield and quality of milk, meat, wool etc.

Express large quantities of foreign proteins in milk, serum or blood of animals. Animals are referred as bioreactors and the approach is called molecular farming or gene farming

To correct the function of nonfunctional genes (causing genetic disorders) by introducing normal or functional copies of genes, referred as gene therapy

Creation of specific cell lines or transgenic animal deleted with known gene to study its importance in development process. Such animals are called Knock out

Transfection Methods

Calcium phosphate precipitationDimethylaminoethyl-dextran) DEAE-dextran

mediated transfectionUptake DNA by endocytosis of complex of DNA and

polycationic

LipofectionUsing liposomes

Fusion with bacterial protoplast with cultured cells with PEG

ElectroporationMicroinjectionViral vectors

Vector systems used for transfection

Viral vectorsPapova virus

SV40 virusSV40 transducing vectorsLate replacement vectorsEarly replacement vectorsSV40 plasmid vectorsNon replicating vectors (passive transfecting

vectors)

Retro VirusesVaccinia virusesAdeno VirusesBaculo viruses (For Transfecting insect cell line) or P

Element vectors

Mammalian Artificial chromosomeBaculo viruses (for expression in insect cell line)

Mammalian Cell lines expression systems

•Two modes of expression - transient and stable.

•Cell lines used. Three cell types are dominant in transient expression: human embryonic kidney (HEK), COS and baby hamster kidney (BHK), whilst CHO (Chinese hamster ovary) cells are used predominantly for stable expression.

•Mammalian expression vectors. Eukaryotic origin of replication is from an animal virus: e.g. Simian virus 40 (SV40). Popular markers for selection are the bacterial gene Neor (encodes neomycin phosphotransferase), which confers resistance to G418 (Geneticin), and the gene, encoding dihydropholate reductase (DHFR). When DHFR is used, the recipient cells must have a defective DHFR gene, which makes them unable to grow in the presence of methotrexate (MTX), unlike transfected cells with a functional DHFR gene. Promoter sequences that drive expression of both marker and cloned heterologous gene, and the transcription termination (polyadenilation signals) are usually from animal viruses (human CMV, SV40, herpes simplex virus) or mammalian genes (bovine growth hormone, thymidine kinase).

•Strategies for co-expression of two cloned genes.

Expression Vectors for Mammalian Cells

Sometimes required for difficult-toexpressproteins or for “completeauthenticity” (matching glycosylation andsequence)• Cells are typically derived from theChinese Hamster Ovary (CHO) cell line• Vectors usually use SV-40 virus, CMV orvaccinia virus promoters and DHFR(dihydrofolate reductase) as the selectable

marker gene

Mammalian Expression

• Gene initially cloned and plasmid propagated in bacterial cells

• Mammalian cells transformed by electroporation (with linear plasmid) and gene integrates (1 or more times) into randomlocations within different CHO chromosomes

• Multiple rounds of growth and selection using methotrexate to select for those cells withhighest expression & integration of DHFR and the gene of interest

Expression System Selection

• Choice depends on size and character ofprotein– Large proteins (>100 kD)? Choose eukaryote– Small proteins (<30 kD)? Choose prokaryote– Glycosylation essential? Choose baculovirusor mammalian cell culture– High yields, low cost? Choose E. coli– Post-translational modifications essential?Choose yeast, baculovirus or other eukaryote

Mammalian Systems

Disadvantages

Selection takes time

(weeks for set-up)

Cell culture is only

sustainable for limited

period of time Set-up is very time

consuming, costly, modest yields

Advantages

Can express large

proteins (>50 kD)

Correct glycosylation

& signal peptide

removal, generates

authentic proteins

Has chaperonins to

help fold “tough” prtns

pcDNA1.1

The vector pcDNA1.1/Amp contains the SV40 and polyoma origins, a transcription unit comprising the human cytomegalovirus promoter and SV40 intron/ polyadenylaton site, an interstitial polylinker to insert the transgene and the ampicillin-resistance marker for selection in E. coli

• 1997 – first eukaryotic organism sequenced• 6607 ORF’s (see below)• Saccharomyces Genome Database

Saccharomyces cerevisiae as a MODEL SYSTEM

As of Aug 12, 2009

   4821 ORFs, 72.97%    975 ORFs, 14.76%    811 ORFs, 12.27%

Yeast systems for heterologous expression: Saccharomyces cerevisiae

Eukaryote, unicellular, GRAS (Generally Regarded As Safe), capable of performing post-translational modifications. Excellent recombinant technology: vectors, markers, methods for transformation and gene manipulation, homologous recombination of cloned sequences by single cross over (insertion) and double cross over

Intracellular expression - higher protein yields, but more difficult extraction and purification. Additional potential problems with:a/ co- and post-translational processing of proteins at N- and C-termini. b/ proteolytic degradation c/ addition of tags might result in aggregation and insolubility

SecretionThe yeast secretory pathway is very similar to that in higher eukaryotes.N-terminal signal sequences for co-translational translocation of screted proteins into the ER are removed by a signal peptidase. Examples of popular signal sequences used for secretion of heterologous proteins -these of Pho5, Suc2 and the a -factor. Modification by N-linked (to asparagine) and O-linked (to serine/threonine) glycosylation. Hyperglycosylation (outer chain extension) in the yeast Golgi is not typical of mammalian cells. Yeast proteins only modified by mannosylation (no other sugars).

Continue….Specific problems with secretion of heterologous proteins

Hyperglycosylation can inhibit reactivity with AB, or render proteins immunogenic (a problem for the production of therapeutic glycoproteins). The obvious solutions: glycosylation mutants (mnn1, mnn9) or elimination of potential sites for glycosylation. Alternatively use other yeast species like P. pastoris.

The cell wall permeability can be a limiting factor. Some cell wall mutants have higher cell wall porosity and release, as a result, heterologous proteins better.

Folding of secreted proteins in the ER and involves accessory proteins such as BiP (the product of KAR2), and PDI (protein disulphide isomerase). Overexpression of these genes has been beneficial in some cases.

Proteolytic processing could be limited by insufficient amounts of required processing enzymes, and in particular the products of SEC11, KEX2, STE13 and KEX1 in cases of multicopy expression of proteins. Again might need to overexpress some of these genes.

Baculovirus

• Baculovirus are present in invertebrates primarily insect species

• They are not infectious for vertebrates & plants• Genome is covalently closed circular double stranded of 134

kbp, due to its small it can accommodate large fragments of foreign DNA

• They are divided into two groups on the basis of their structure as-:

Nucleopolyhedroviruses (NPV) Granuloviruses These NPV are mainly used as expression vectors i.e.

Autographa californica NPV (AcMNPV) isolated from the larva of the alfalfa looper

Contd..

• Baculovirus expression system based upon the ability to propagate AcMNPV in insect cells

• Uses many of the protein modification, processing• and transport systems present in higher eukaryotic• cells.• Virus that can be propagated to high titers adapted• for growth in suspension cultures• obtain large amounts of recombinant protein with• relative ease• Baculovirus are noninfectious to vertebrates and• their promoters are inactive in mammalian cells.

Baculovirus expression system

• Recombinant baculovirus have become widely used as vectors to express heterologous genes in cultured insect cells and insects larvae

• Heterologous genes placed under the transcriptional control of the strong polyhedrin promoter of the Autographa californica polyhedrosis virus (AcNPV)

• Based on site specific transposition of an expression cassette (pfast Bac with gene of interest) into a baculovirus shuttle vector (bacmid)

Steps in recombinant baculovirus production

• Clone the gene of interest in pfast Bac donor plasmid• Expression cassette in pfast Bac is flanked by left and right

arms of Tn7 and also an SV40 polyadenylation signal to form a miniTn7

• Cloned pfast Bac is transformed in E.coli host strain (DH10Bac) which contains a baculovirus shuttle vector bacmid having a mini-attTn7 target site

• Helper plasmid which allows to transpose the gene of interest from pfast to bacmid (shuttle vector)

• Transposition occurs between the mini-att Tn7 target site to generate a recombinant bacmid

• This recombinant bacmid can now be used to transfect insect cell lines.

Baculovirus Expression

Baculovirus Systems

Disadvantages

Grow very slowly (10-

12 days for set-up)

Cell culture is only

sustainable for 4-5

days

Set-up is time

consuming, not as

simple as yeast

Advantages

Can express large

proteins (>50 kD)

Correct glycosylation

& signal peptide

removal

Has chaperonins to

help fold “tough” prtns

Very high yields, cheap