Expression Vectors

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 of cloned genes in eukaryotes has certain advantages. The most important being the ability of eukaryotic organisms to bring about post-translational modifications—glycosylation, phosphorylation, correct disulfide bond formation, proteolytic cleavage etc. Eukaryotic expression systems produce stable and biologically active proteins.

Expression with Yeast Vector,
The insertion of a foreign DNA into bacterial and yeast cells is referred to as transformation. The term transfection is used for the introduction of a foreign DNA into animal cells. The insert DNA in the eukaryotic cells may be associated with vector or integrated into the host chromosomal DNA.
The common yeast Saccharomyces cerevisiae is widely used as a host for the expression of cloned genes. There are many justifiable reasons for its extensive use.
i. S. cervisiae is single-celled that can be easily grown. Its biochemistry, genetics and physiology are quite known.
ii. It has a naturally occurring plasmid and strong promoters for efficient expression.
iii. S. cerevisiae can bring about many post t¬ranslational changes in proteins.
iv. The secreted recombinant proteins can be easily isolated, since very few host proteins are secreted.
v. The U.S. Food and Drug Administration has certified S. cerevisiae as a generally recognized as a safe (CRAS) organism.
As such, S. cerevisiae has been in use for several decades in baking and brewing industries. Biotechnologists work quite comfortably with this yeast to produce a large number of recombinant proteins. These include insulin, a1 -antitrypsin, hepatitis B virus surface antigen, platelet derived growth factor, fibroblast growth factor and HIV-I antigens. These products are in use as diagnostic agents, vaccines, and therapeutic agents.
Vectors for S. cerevisiae: There are three types of vectors for S. cerevisiae:

1. Episomal or plasmid vectors.
2. Integrating vectors.
3. Yeast artificial chromosomes (YACs).
   Post-translational modifications by S. cerevisiae:
   The heterologous proteins synthesized by S. cerevisiae undergo post-translational changes while they are being exported into the extracellular environment. To facilitate protein secretion, a single (leader) peptide is attached to the protein. This peptide is removed by the yeast endoprotease.

Expression with Baculovirus
Baculovirus are envelope double standard DNA viruses with rod shaped nucleocapsids, the life cycles involves 2 distinct form of viruses budded virus as well as occluded virus. The budded virus consists of a single nucleocapsid enveloped by the protein called GP64 a virus derived like protein, and host membrane proteins, whereas occluded virus consists of multiple nucleocapsids embedded in a protein matrix called as a polyhedrin matrix. The most extensively studied Baculovirus strain is called as the autographa calefornia multiple nuclear polyhedrosis polyhedrosis virus or abbreviated as ACMNP AMD. This ACMNP infects larval lepidopteron, so this is the basic phases of the Baculovirus which is based on which an expression system has been developed for making for recombinant proteins in yeast cells.
Baculo viruses exclusively infect insect cells. The DNA of these viruses encode for several products and their productivity in cells is very high to the extent of more than 10,000 times compared to mammalian cells. Besides carrying a large number of foreign genes, the baculoviruses can effectively express and process the products formed. Another advantage with these viruses is that they cannot infect humans, other vertebrates or plants. Thus, baculoviruses are safe vectors.
The polyhedrin gene is responsible for the synthesis of a matrix protein-polyhedrin. This protein is synthesized in large quantities by baculovirus during the infection cycle. Polyhedrin protects the virus from being inactivated by environmental agents. The promoter for polyhedrin gene is very strong. However, the life-cycle of baculovirus does not depend on the presence of this gene. Polyhedrin gene can be replaced by a cloned gene, and the genetically engineered baculovirus can infect the cultured insect cells.
The cloned gene expresses, and large quantities of recombinant proteins are produced. Because of a close similarity in the post-translational modifications between insects and mammals, biologically active proteins can be produced by this approach. And in fact, by using baculovirus as an expression vector system, a good number of mammalian and viral proteins have been synthesized e. g adenosine deaminase, alkaline phosphatase, malaria proteins etc.
The organization of a baculovirus (AcMNPV) transfer vector is shown below. It consists of an E. coli-based plasmid vector along with the DNA of baculovirus. This in turn has AcMNPV DNA, a polyhedrin promoter region, cloning site for insert DNA and polyhedrin termination region (Fig. 11.3A).
When the insect culture cells, transfected with AcMNPV are mixed with transfer vector carrying a cloned gene, a double crossover occurs. The result is that the cloned gene with polyhedrin promoter and termination sequences gets integrated into AcMNPV DNA (Fig. 11.3B). In this process, polyhedrin gene is lost. The recombinant baculovirus containing cloned gene is isolated.

The host insect culture cells, on infection with recombinant baculovirus, produce heterologous proteins. A large number and a wide variety of recombinant proteins (around 500) have been synthesized in the laboratory. A majority of them (>95%) have the requisite post-translational modifications.
Mammalian Cell Expression Vectors:
Mammalian expression vectors are useful for the production of specific and authentic recombinant proteins (for use as therapeutic agents). In addition, they are also helpful for studying the function and regulation of mammalian genes. In general, the mammalian expression vectors are quite comparable to other eukaryotic expression vectors. . However, large-scale production of recombinant proteins with engineered mammalian cells is costly.
A diagrammatic representation of mammalian vector is shown in the figure below. It contains a eukaryotic origin of replication from an animal virus such as Simian virus 40 (SV40) and a prokaryotic origin of replication (from E. coli). The mammalian vector has a multiple cloning site and a selectable marker gene. Both of them are under the control of eukaryotic promoter and polyadenylation sequences.

These sequences are obtained from either animal viruses (SV40, herpes simplex virus) or mammalian genes (growth hormone, metallothionein). The promoter sequences facilitate the transcription of cloned genes (at the multiple cloning site) and the selectable marker genes. On the other hand, the polyadenylation sequences terminate the transcription. Ampicillin resistant marker gene can be used for selecting the transformed E. coli cells.