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 to study 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 of cloned genes. Promoter strength Transcriptional termination Plasmid copy number Plasmid stability Host-cell physiology Translational initiation sequences Codon choice mRNA 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 g/mg total protein. Vectors with strong, controllable promoters are 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 promoters Are 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 promoter melon 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 are cytomegalovirus immediate-early gene promoter (CMV) human desmin (Des) human alpha-myosin heavy chain (-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 precipitation Dimethylaminoethyl-dextran) DEAE-dextran mediated transfection Uptake DNA by endocytosis of complex of DNA and polycationic Lipofection Using liposomes Fusion with bacterial protoplast with cultured cells with PEG Electroporation Microinjection Viral vectors Vector systems used for transfection Viral vectors Papova virus SV40 virus SV40 transducing vectors Late replacement vectors Early replacement vectors SV40 plasmid vectors Non replicating vectors (passive transfecting vectors) Retro Viruses Vaccinia viruses Adeno Viruses Baculo viruses (For Transfecting insect cell line) or P Element vectors Mammalian Artificial chromosome Baculo 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-toexpress proteins or for complete authenticity (matching glycosylation and sequence) Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line Vectors usually use SV-40 virus, CMV or vaccinia 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 random locations 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 of protein Large proteins (>100 kD)? Choose eukaryote Small 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 ORFs (see below) Saccharomyces Genome Database Saccharomyces cerevisiae as a MODEL SYSTEM As of Aug 12, 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 Secretion The 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 ( 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