Gene expression vector by tahura mariyam ansari

this is a presentation on gene expression vector that includes what is expression vector, how many types of expression vector and difference between cloning and expression vector

1. TOPIC- GENE EXPRESSION VECTOR PRESENTED BY: TAHURA MARIYAM MSc. MICROBIOLOGY PRENENTED TO: Dr. Gurudayal Ram (Assistant prof. Sr. Grade) DEPARTMENT OF INDUSTRIAL MICROBIOLOGY JACOB INSTITUTE OF BIOTECHNOLOGY AND BIO-ENGINEERING SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE,TECHNOLOGY, AND SCIENCES, PRAYAGRAJ

2. C ontent INTRODUCTION ELEMENTS OF EXPRESSION VECTOR ELEMENTS OF EXPRESSION PROTEIN TAGS OTHERS EXPRESSION/PRODUCTION VECTOR BACTERIA YEAST CELL-FREE SYSTEM APPLICATION PRODUCTION OF PEPTIDE AND PROTEIN PHARMACEUTICALS

3. Introduction An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins .   The vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of significant amount of stable messenger RNA, which can then be translated into protein. The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity when necessary through the use of an inducer, in some systems however the protein may be expressed constitutively. Escherichia coli is commonly used as the host for protein production, but other cell types may also be used. An example of the use of expression vector is the production of insulin, which is used for medical treatments of diabetes.

4. Expression Vector The cloning vector containing suitable expression signals to have maximum gene for expression is called expression vector. The following expression signals are introduced into gene cloned vectors are get maximum expression Insertion of strong promoter. Insertion of strong termination codon. Adjustment of distance between promoter and cloned gene. Insertion of transcription termination sequence.

5. Choice of expression vector Strong promoter Intact ORF Ribosomal binding site Termination sequence

6. Expression in Prokaryotes Promoter- commonly used inducible promoter are promoters derived from lac operon and the T7 promoter. Other strong promoters used include Trp promoter and Tac promoter, which a hybrid of both the Trp and Lac operon promoters Ribosome binding site(RBS) following the promoter, and promoter efficient translation of the protein of interest. Translation initiation site- shine dalgarno sequence enclosed in the RBS,8 base-pair upstream of the AUG start codon.

7. Post-Translational Modification Eukaryotic protein synthesis occurs in cytoplasm or on the endoplasmic reticulum . These protein are further are further post translation processed that is required for protein activity and stability. Disulfide isomerase also make sure that the proteins produced have the correct configuration The proper glycosylation that are necessary for protien conformation, localization by interacting with specific receptor and increase stability.

8. Eukaryotic E xpression System Eukaryote expression vectors require sequence that encode for. Polyadenylation tail : creates a polyadenylation tail at the end of the transcribed pre-mRNA that protects mRNA from exonucleases and ensures transcriptional termination: stabilizers mRNA production. Minimal UTR length: UTRs contains a specific characteristics that may impede transcriptional or translation, and thus the shortest UTRs or none at are encoded for in optimal expression vectors. Kozak sequence: vectors should encode for a Kozak sequence in the m RNA, which assembles the ribosome for translation of the m RNA.

9. Eukaryotic Expression System contd…. Eukaryote expression vector require sequence that encode for. Polyadenylation tail: creates a polyadenylation tail at the end of the transcribed pre-m RNA that protects the m RNA fromexonucleases and ensure transcriptional and translational termination: stabilizers m RNA production. Minimal UTR length: UTRs contains specific characteristics that may impede transcription or translation, and thus the shortest UTRs or none at all are encoded for in optimal expression vectors. Kozak sequence: vectors should encode encode for kozak sequence in the m RNA, which assembles the ribosome for translation of the m RNA.

10. Element of of expression vector An expression vector has features that any vector may have, such as an origin of replication, a selectable marker, and a suitable site for the insertion of a gene like the multiple cloning site. The cloned gene may be transferred from a specialized cloning vector to an expression vector, although it is possible to clone directly into an expression vector. The cloning process is normally performed in Escherichia coli. Vectors used for protein production in organisms other than E.coli may have, in addition to a suitable origin of replication for its propagation in E. coli, elements that allow them to be maintained in another organism, and these vectors are called shuttle vectors.

11. The major feature of a eukaryotic expression vector are a promoter, a multiple cloning site, DNA segment for termination and polyadenylation, selectable marker, original of replication in E.coli and eukaryotic cell and Ampr for marker in E.coli

12. Eukaryotic Expression System Saccharomyces cerevisiae Pichia pastoris Baculovirus-insect cell lines Mammalian systems

13. Elements of Expression Vector contd…. An expression vector must have elements necessary for gene expression. These may include a promoter, the correct translation initiation sequence such as a ribosomal binding site and start codon, a termination codon, and a transcription termination sequence. There are differences in the machinery for protein synthesis between prokaryotes and eukaryotes, therefore the expression vectors must have the elements for expression that are appropriate for the chosen host. For example, prokaryotes expression vectors would have a Shine- Dalgarno sequence at its translation initiation site for the binding of ribosomes, while eukaryotes expression vectors would contain the Kozak consensus sequence .   The promoter initiates the transcription and is therefore the point of control for the expression of the cloned gene. The promoters used in expression vector are normally inducible, meaning that protein synthesis is only initiated when required by the introduction of an inducer such as IPTG. Gene expression however may also be constitutive (i.e. protein is constantly expressed) in some expression vectors. Low level of constitutive protein synthesis may occur even in expression vectors with tightly controlled promoters.

14. Protein Tags After the expression of the gene product, it is usually necessary to purify the expressed protein; however, separating the protein of interest from the great majority of proteins of the host cell can be a protracted process. To make this purification process easier, a purification tag may be added to the cloned gene. This tag could be histidine (His) tag, other marker peptides, or a fusion partners such as glutathione S-transferase or maltose-binding protein. Some of these fusion partners may also help to increase the solubility of some expressed proteins. Other fusion proteins such as green fluorescent protein may act as a reporter gene for the identification of successful cloned genes, or they may be used to study protein expression in cellular imaging. OTHERS : The expression vector is transformed or transfected into the host cell for protein synthesis. Some expression vectors may have elements for transformation or the insertion of DNA into the host chromosome, for example the vir genes for plant transformation, and integrase sites for chromosomal integration. Some vectors may include targeting sequence that may target the expressed protein to a specific location such as the periplasmic space of bacteria.

15. Expression/Production System Different organisms may be used to express a gene's target protein, and the expression vector used will therefore have elements specific for use in the particular organism. The most commonly used organism for protein production is the bacterium Escherichia coli. However, not all proteins can be successfully expressed in E. coli, or be expressed with the correct form of post-translational modifications such as glycosylations, and other systems may therefore be used .  

16. Bacteria The expression host of choice for the expression of many proteins is Escherichia coli as the production of heterologous protein in E. coli is relatively simple and convenient, as well as being rapid and cheap. A large number of E. coli expression plasmids are also available for a wide variety of needs. Other bacteria used for protein production include Bacillus subtilis .   Most heterologous proteins are expressed in the cytoplasm of E. coli. However, not all proteins formed may be soluble in the cytoplasm, and incorrectly folded proteins formed in cytoplasm can form insoluble aggregates called inclusion bodies. Such insoluble proteins will require refolding, which can be an involved process and may not necessarily produce high yield

17. FIG- EXPRESSION VECTOR

18. Proteins which have disulphide bonds are often not able to fold correctly due to the reducing environment in the cytoplasm which prevents such bond formation, and a possible solution is to target the protein to the periplasmic space by the use of an N-terminal signal sequence.   Another possibility is to manipulate the redox environment of the cytoplasm. Other more sophisticated systems are also being developed; such systems may allow for the expression of proteins previously thought impossible in E. coli, such as glycosylated proteins . The promoters used for these vector are usually based on the promoter of the lac operon or the T7 promoter, and they are normally regulated by the lac operator. These promoters may also be hybrids of different promoters, for example, the Tac-Promoter is a hybrid of trp and lac promoters. Note that most commonly used lac or lac-derived promoters are based on the lacUV5 mutant which is insensitive to catabolite repression. This mutant allows for expression of protein under the control of the lac promoter when the growth medium contains glucose since glucose would inhibit gene expression if wild-type lac promoter is used. Presence of glucose nevertheless may still be used to reduce background expression through residual inhibition in some systems . Examples of E. coli expression vectors are the pGEX series of vectors where glutathione S-transferase is used as a fusion partner and gene expression is under the control of the tac promoter, and the pET series of vectors which uses a T7 promoter.

19. Bacteria Contd… It is possible to simultaneously express two or more different proteins in E. coli using different plasmids. However, when 2 or more plasmids are used, each plasmid needs to use a different antibiotic selection as well as a different origin of replication, otherwise one of the plasmids may not be stably maintained. Many commonly used plasmids are based on the ColE1 replicon and are therefore incompatible with each other; in order for a ColE1-based plasmid to coexist with another in the same cell, the other would need to be of a different replicon, e.g. a p15A replicon-based plasmid such as the pACYC series of plasmids. Another approach would be to use a single two- cistron vector or design the coding sequences in tandem as a bi- or poly- cistronic construct.

20. Yeast A yeast commonly used for protein production is Pichia pastoris. Examples of yeast expression vector in Pichia are the pPIC series of vectors, and these vectors use the AOX1 promoter which is inducible with methanol. The plasmids may contain elements for insertion of foreign DNA into the yeast genome and signal sequence for the secretion of expressed protein. Proteins with disulphide bonds and glycosylation can be efficiently produced in yeast. Another yeast used for protein production is Kluyveromyces lactis and the gene is expressed, driven by a variant of the strong lactase LAC4 promoter.   Saccharomyces cerevisiae is particularly widely used for gene expression studies in yeast, for example in yeast two-hybrid system for the study of protein-protein interaction. The vectors used in yeast two-hybrid system contain fusion partners for two cloned genes that allow the transcription of a reporter gene when there is interaction between the two proteins expressed from the cloned genes. Baculovirus is normally used for production of glycoproteins, although the glycosylations may be different from those found in vertebrates. In general, it is safer to use than mammalian virus as it has a limited host range and does not infect vertebrates without modifications.

21. YAC Cloning System A YAC is designed to clone a large segments of DNA (100kb), which is then maintained as a separate chromosome in the host yeast cell. It is highly stable and has been used for the physical mapping of human genomic libraries . It has a sequences that act as ARS for replication, centromere for cell division, and telomere for stability. To date, they have not been used as expression systems for the commercial production.

22. Intercellular Production in Yeast Human Cu/ZnSOD c DNA was cloned between the promoter and termination-polyadenylation sequence of the yeast GAPD gene and subsequently used to transform LEU mutant host cell.

23. Secretion of Heterologous Proteins Proteins may also be production for secretion. In this system, any glycosylated protein is selected The coding sequence of recombinant protein must be cloned downstream of a leader sequence, the appropriate posttranslational modifications occur, and an active recombinant Protein in secreted. The leader peptide is removed by endoprotease that recognizes the Lys-Arg. For Example-a properly processed and activity from the protein hirudine; coding sequence to allow expression that is cleaved away in processing. Producing a recombinant in yeast rather than in bacteria is to ensure the proper folding.

24. Difference Between Cloning Vector & Expression Vector

25. Secretion of heterologous proteins

26. Cell- Free System E. coli cell lysate containing the cellular components required for transcription and translation are used in this in vitro method of protein production. The advantage of such system is that protein may be produced much faster than those produced in vivo since it does not require time to culture the cells, but it is also more expensive. Vectors used for E. coli expression can be used in this system although specifically designed vectors for this system are also available. Eukaryotic cell extracts may also be used in other cell-free systems, for example, the wheat germ cell-free expression systems. Mammalian cell-free systems have also been produced.

27. Vector Design Generalized mammalian expression vector . The MCS and SMG are under the control of eukaryotic promoter, polyadenylation, and terminal sequences. An intron enhance the production of heterologous protein. The AMPr gene is used for selecting transformed E.COLI. For the best results, a gene of interest must be equipped with translation control sequence A gene of interest with a various sequences that enhance translation and facilitate both secretion and purification. A Kozak sequence, specific sequence surrounding the AUG start codon, signal sequence, protein affinity tag for purification, protelaolytic cleavage site,and stop codon. The 5’ & 3’ UTR increase the efficiency of translation and contribute to mRNA stability.

28. Two Vector Expression System

29. Two Vector Expression System contd….

30. Application LABORATORY Expression vector in an expression host is now the usual method used in laboratories to produce proteins for research. Most proteins are produced in E. coli, but for glycosylated proteins and those with disulphide bonds, yeast, Baculovirus and mammalian systems may be used.

31. Production of Peptide and Protein Pharmaceuticals Most protein pharmaceuticals are now produced through recombinant DNA technology using expression vectors. These peptide and protein pharmaceuticals may be hormones, vaccines, antibiotics, antibodies, and enzymes. The first human recombinant protein used for disease management, insulin, was introduced in 1982. Biotechnology allows these peptide and protein pharmaceuticals, some of which were previously rare or difficult to obtain, to be produced in large quantity. It also reduces the risks of contaminants such as host viruses, toxins and prions. Examples from the past include prion contamination in growth hormone extracted from pituitary glands harvested from human cadavers, which caused Creutzfeldt– Jakob disease in patients receiving treatment for dwarfism, and viral contaminants in clotting factor VIII isolated from human blood that resulted in the transmission of viral diseases such as hepatitis and AIDS. Such risk is reduced or removed completely when the proteins are produced in non-human host cells .

32. Reference https :// www.cusabio.com/c-20273.html https://en.wikipedia.org/wiki/Expression_vector