Expression vectors

Expression vectors express the desired gene within the host. The gene of interest gets inserted into the vector so that it encodes a protein or a peptide. Both the structural genes and promoter sequences get cloned into the same segment of DNA. If a gene gets cloned into a vector, it may or may not express. However, cloning the structural genes with the promoter sequence and then inserting them into the vector helps in the expression of the desired gene. Thus an expression vector consists of a cloned gene, sequences upstream of the cloned genes, and the genes controlling the transcription and translation. The blue-white screening technique helps to check whether the gene gets expressed or not. The expression construct involves a plasmid or a virus designed for gene expressions. Common features of an expression vector involve the origin of replication, a selectable marker, and a suitable insertion site known as multiple cloning sites. It is also known as a polylinker.

Hence, a polylinker is a common site for many restriction enzymes. It gets cleaved by any of the restriction enzymes. The DNA fragment of interest gets inserted in place of the cleaved restriction sites. The gene expression elements present in the expression vector play an important role.

The gene expression elements include a promoter, a translation initiation sequence, a ribosome binding site, a start codon, a stop codon, and translation termination sequences. The expression vector gets designed as per the type of the host. Prokaryotic expression vector consists of sequences suitable for a prokaryotic host. Eukaryotic expression vectors consist of sequences suitable for a eukaryotic host. Shine-Dalgarno sequence belongs to a prokaryotic expression vector. Kozak sequence belongs to a eukaryotic expression vector. The protein purification follows the expression of the genes through an expression vector. Hence, protein purification plays an important role in obtaining the purified product. A purification tag such as a histidine tag, a marker peptide or a fusion partner gets added to a cloned gene. Let us know the examples of the expression vectors in detail.

1. pGEX-3X vector

It is an E. Coli expression vector. The fusion partner involved in this vector is known as glutathione-S-transferase. The gene expression gets controlled through the lac promoter. The pGEX-3X is a 4952 base pair expression vector. It consists of a protein binding site, a lac repressor, a multiple cloning sites, a bla promoter, and a beta-lactamase site. It also consists of the ampicillin resistance gene. The GST enzyme family corresponds to many types of proteins such as the cytosolic, mitochondrial, and microsomal proteins. The expression of the fusion protein gets regulated under the control of the lac promoter. Addition of IPTG induces the protein expression. The GST fusion protein exhibits an ability to bind to a glutathione agarose column. After washing away the unwanted protein, the fusion protein remains as it is. The yield includes 1-10 mg of fusion protein per liter of cells.

 Image: The Expression Vector (pGEX-3X)

2. pMal expression vectors:

It employs a fusion protein known as maltose binding protein (MBP). The lac promoter controls the expression system. The purification of the expression vector involves affinity chromatography on an amylose agarose column. The pMal expression vectors consist of multiple cloning sites and encode oligopeptide proteolytic cleavage site. The vector yields about 5-30 mg of the protein.

3. pET-41a (+) vector:

It consists of a kanamycin resistance gene as a selectable marker. The fusion tag used in this vector is known as glutathione-S-transferase (GST) gene. Unlike other expression vectors, pET-41 a(+) does not have a stop codon after the GST gene. T7 promoter controls the expression of the GST gene and the fusion gene. IPTG (isopropyl -beta-D-thiogalactopyranoside) induces protein expression.

4. Eukaryotic expression vector (pcDNA3):

Introduction of eukaryotic expression plasmid into the cells enables a stable expression. Eukaryotic expression vector requires a specific eukaryotic sequence. A eukaryotic expression vector constitutes three main components such as an ATG codon, a Kozak sequence, and a polyadenylation signal sequence. Transfection process involves electroporation or a calcium chloride treatment.

Applications of expression vectors:

Peptides or proteins get expressed through the expression vector. Recombinant DNA technology helps to derive most of the proteins in the pharmaceutical industry. Hence, they are known as recombinant proteins. Examples include hormones, peptide vaccines, antibiotics, enzymes, antibodies, and protein-based factors. The first human recombinant protein is known as insulin. Screening of cDNA sequences involves expression vectors transformed into bacterial cells grown on a selective medium. Detection of protein-protein interactions involves expression vectors. 
Yeast two-hybrid system is an example of protein-protein interaction. A yeast expression consists of a sequence for Gal4p binding domain fused to a sequence coding a protein. The experiment involves the cotransformation of yeast expression vector with yeast expression library into the yeast cells. Here the fusion protein gets synthesized.

Working model of yeast expression vector:

It involves a yeast galactose metabolism gene GAL1 transcription. The gal4 gene encodes gal4p regulatory protein. The gal4p binds to an upstream activator sequence G. It is an example of a promoter element. Two domains of gal4p include DNA binding domain and an activation domain. The DNA binding domain BD binds to an upstream activator sequence G. An activation domain helps in binding the RNA polymerase to a promoter region. Hence it helps in the initiation of transcription.

Types of expression vectors used in a two-hybrid model:

One type of an expression vector contains gal4p BD sequence fused with a known sequence encoding a protein. The other type of expression vector consists of gal4pAD fused with protein-coding cDNA sequence. Cotransformation of AD and BD plasmids further involve the study of the reporter gene expression.

Production of protein product using an expression vector:

A classic example of recombinant protein production involves a transgenic sheep. An expression vector consisting of a gene of interest must be suitable for the mammalian cells. The beta-lactoglobulin promoter sequence involves the expression of the protein. The protein product isolation becomes simple through milk secretion. The beta-lactoglobulin promoter is an active mammary tissue promoter.

Manipulation of the gene of interest enables its placement adjacent to the beta-lactoglobulin promoter. Hence the expression vectors now get microinjected into the sheep ova. Each ova having the expression vector gets implanted into the foster mother. Identification of transgenic sheep involves PCR analysis. PCR easily detects recombinant sequence. After maturation of the transgenic sheep, the beta-lactoglobulin gets expressed in the mammary tissue. It leads to the production of milk. Separation techniques separate the proteins from the milk.

Other examples of applications of the expression vectors:

·  Human growth hormone production is involved in the pituitary dwarfism treatment

·        Bovine growth hormone increases the cattle and the dairy yields.

References:
[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2] Genetics, 9th Edition (Multicolour Edition), Verma P.S. & Agarwal V.K.

[3] Gene cloning and DNA analysis, T.A. Brown

[4] Genetic Engineering, Verma P.S. & Agarwal V.K.
[5] Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh

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