Expression vector, baculovirus expression vector
- 1. Expression Vector, Baculovirus Expression vector Promila Sheoran Ph.D. Biotechnology GJU S&T Hisar
- 2. Expression Vector •The knowledge that expression of protein coding gene can be induced by placing this gene downstream of a promoter has led to the development of a number of expression vectors, both prokaryotic as well as eukaryotic. • So, today, it is possible if we want, for example, any gene that you are interested in, whether this gene can code for insulin or growth hormone or any protein that is of interest to you, you can take this gene, put it downstream of promoter of your choice, and you can make this protein in the particular organism of your choice.
- 3. •For example, suppose you want to express whether insulin or growth hormone or hepatitis B surface antigen or a clotting factor, you simply take these genes which code for this respective proteins and clone into a promoter of your choice. •For example, if you want to express this in a bacterial system, you put this gene in front of a bacterial promoter, and you have to construct what is called as a bacterial expression plasmid. •On the other hand, if you want to express this gene in yeast cells, you have to put this gene in front of yeast promoter and construct what is called as yeast expression vector. •So, you can see, the idea or the knowledge that promoters, which contain binding for transcription factor sites and RNA polymerase, they can be exploited for expression. The expression of any downstream gene has now let to the development of what are called as a recombinant DNA technology and production of recombinant proteins of your choice.
- 4. So, what is an expression vector? •An expression vector is usually a plasmid that is used to introduce a specific gene into a target cell, and express the protein that is coded for by the gene. •So, once inside the host cell, the gene encoded by the expression vector is transcribed by the host transcription machinery; that is, the host transcription factor, • and host RNA polymerase will transcribe the gene, and the RNA that is synthesized is then translated by the host translation machinery, leading to the synthesis of a particular protein of your interest.
- 5. •So, if the gene has to be expressed inside the host cells, the expression plasmid should contain regulatory sequences that act as a either enhancer and promoter regions, and lead to efficient transcription of genes carried by the expression vector. •So, if you want to express a gene of your interest in a particular system, either bacteria or yeast or mammalian system, the regulator region that you have chosen must contain a promoter and powerful enhancers, • so that powerful transcription factors can go and bind to this sequences and a large amount of mRNA can be synthesized, which in turn, gets translated into a protein, and you can then make a protein of your interest in large amounts. •So, the goal of a well-designed expression vector is, therefore, production of large amounts of mRNA, and therefore, large amounts of proteins. So, this is what is the rationale behind constructing an expression vector.
- 6. •So, usually, expression vectors contain a very strong promoter upstream of the cloned gene, as well as a strong terminator promoter. • Phage gene promoters such as lambda leftward promoter or a T7 promoter are very popularly used in the vectors like pET vectors and pT7 vectors, which are very, very popular expression vectors that people now use for making proteins. •T7 promoter-based expression vectors, are extensively used today in the area of molecular biology and recombinant DNA technology.
- 7. Mechanism of T7 expression system •T7 RNA polymerase is a RNA polymerase coded by, required by the T7 bacteriophage, and this T7 RNA polymerase recognize a very short sequence, about 15 to 20 bases. It is called as a T7 promoter. •So, what you do is that you place this T7 RNA polymerase gene under the promoter– under a IPTG inducible lac promoter– • and put it in a chromosomal DNA of a bacterial cell, and usually, the E.coli strain which harbors such a T7 RNA polymerase in the bacterial chromosome and is usually called BL21 DE3 E. coli cells. • Then, you introduce your plasmid, which actually contains your gene of interest downstream of a T7 promoter.
- 8. Transcription initiates with the binding of RNA polymerase to the promoter sequence. Prokaryotic expression systems makes use of the strongest promoter like T5 E.coli RNA polymerase and T7bacteriophagel polymerase. The promoter sequences in most of the plasmids are in control of Lac operon which is repressed by Lac repressor and does not transcribe until the addition of IPTG (Isoproply β-D-thiogalactoside). Hence the promoter sequences are used in regulation of gene expression. The pET expression system best exemplifies such precisely regulated expression system.The pET vector system utilizes host cell whose genome is constructed to contain gene 1 which encodes for T7 polymerase and this gene is under the control of Lac repressor. Further, two plasmids pET and pLysS are inserted into the cell. pET contains T7 promoter, Lac operator followed target gene. PLysS produces T7 lysozyme which inactivates T7 RNA polymerase. Upon the addition of IPTG, the Lac repressor protein is taken away and the transcription proceeds with the help of T7 RNA polymerase. The overall scheme is depicted in figure.
- 9. •So, your foreign gene of interest downstream of the T7 promoter is placed in a plasmid, and then introduce this plasmid into these E.coli cells. •Now, if you take the cells and add IPTG, the IPTG will induce the lac promoter. •The T7 RNA polymerase enzymes synthesized from the bacterial chromosome– that T7 RNA polymerase will now come and bind to the T7 RNA promoter binding promoter site present in the plasmid, and induce the expression of your foreign gene. •So, you can see, the RNA polymerase comes from the bacterial chromosome and goes and binds to the promoter binding site present in the plasmid, and your protein is expressed in very high amounts. • So, the T7 RNA polymerase gene is from T7 phage; it is not present in the E.coli. The T7 RNA polymerase gene is integrated into the chromosome of E.coli using a temperate phage DE3, so DE3 stands for a temperate phage.
- 10. •The T7 RNA polymerase is under the control of a lac promoter. •Therefore, by adding IPTG, you can induce the host to produce the T7 RNA polymerase, and this T7 RNA polymerase activity is much higher than E.coli RNA polymerase, and therefore, protein expression by T7 expression vector is very high. • So, you can express proteins at very high levels using this T7 expression system
- 11. Expression cassette •An expression cassette is a part of a vector DNA used for cloning and transformation. • In each successful transformation, the expression cassette directs the cell's machinery to make RNA and protein. Some expression cassettes are designed for modular cloning of protein-encoding sequences so that the same cassette can easily be altered to make different proteins. •An expression cassette is composed of one or more genes and the sequences controlling their expression. • Three components comprise an expression cassette: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site. •Different expression cassettes can be transformed into different organisms including bacteria, yeast, plants, and mammalian cells as long as the correct regulatory sequences are used.
- 12. Baculovirus Expression Vectors •Baculoviruses are a diverse group of insect-specific viruses, predominantly infecting insect larvae of the order Lepidoptera. By far the most widely studied member of this family is Autographa californica nucleopolyhedrovirus (AcMNPV). •AcMNPV has a circular, double-stranded, super-coiled DNA genome of approximately 130 kilobases packaged in a rod-shaped nucleocapsid. •The virus genome can effectively accommodate large insertions of foreign DNA. • Such insertions of foreign genes into the AcMNPV genome has resulted in production of baculovirus expression vectors; recombinant viruses genetically modified to contain a foreign gene of interest, which can be expressed in insect cells under the control of a baculovirus gene promoter.
- 13. Baculovirus expression vector systems The majority of baculovirus expression systems exploit the polh promoter to drive high level production of foreign proteins. • The baculovirus polh gene is non-essential for virus replication in insect cell cultures and therefore can be removed from the virus genome with no detrimental effect to budded virus production.
- 14. •As the baculovirus genome is generally considered too large for direct insertion of foreign gene, the gene of interest is first cloned into a transfer vector containing sequences that flank the polh gene in the viral genome. • Virus DNA and transfer vector are co-transfected into the host insect cell and homologous recombination between the flanking sequences common to both DNA molecules occurs. •This causes the insertion of the gene of interest into the viral genome at the polh locus, resulting in the production of a recombinant virus genome. •The genome then undergoes replication within the host nucleus, generating recombinant BV containing the foreign gene under the control of the strong, late viral polyhedrin promoter. •Polyhedra are not produced as the polh gene is no longer functional, having been replaced by the gene of interest
- 15. • To improve the efficacy of recombinant virus production, a unique Bsu361 restriction enzyme site was engineered into the polh locus of baculovirus DNA to permit linearization of the viral genome prior to co-transfection, giving rise to a higher frequency of recombinant virus production. Further improvements employed multiple Bsu361 sites, with digestion of the viral DNA resulting in a partial deletion within an essential gene, ORF1629. •The deletion within this gene prevents replication of parental virus, increasing the yield of recombinant virus to more than 90%. Insertion of the Escherichia coli (E. coli) lacZ gene into the polh locus, replacing the polh gene, produced the commercially available BacPAK6.
- 16. A Bsu361 restriction enzyme site is located within the lacZ gene along with 2 additional sites situated in the two flanking genes either side of lacZ. Digestion of BacPAK6 with Bsu361 removes the lacZ gene and a fragment of ORF1629, resulting in linear virus DNA incapable of replicating within insect cells. Co-transfection of insect cells with BacPAK6 DNA and a transfer vector containing the gene of interest restores the deletion in ORF1629 and recircularises the virus DNA by allelic replacement. This restoration of the essential virus gene permits replication within insect cells, followed by assembly of nucleocapsids within the nucleus and ultimately production of recombinant viruses. •The presence of lacZ allows the selection of colourless, recombinant virus plaques against a background of parental, blue plaques in the presence of X-gal.
- 17. •Recombinant baculoviruses have become a widely used system for the production of recombinant proteins within insect cells. • The availability of the entire baculovirus sequence has and will continue to enable further manipulation of the virus genome to increase and further optimise recombinant protein expression in both insect and mammalian cell lines. • It is hoped that the use of recombinant baculoviruses as gene delivery vectors for higher eukaryotic cell lines will become as routine as the use of such viruses for recombinant protein expression within insect cells and that advances in knowledge and technology will continue to expand the possibilities and applications of the baculovirus expression system.
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