1-3.8.2 Examples of Expression Vector:

1-3.8.2.a pET vector:

• pET vector system is a cloning and expression vector system for recombinant protein production in E.coli . This product is registered under trademark of Novagen Inc.
  
  
  •  The original pET vector system was constructed by Studier and colleagues. That plasmid is developed at Novagen with enhanced characteristics.
  
  
  •  Target genes are cloned under strong T7 bacteriophage promoter.
  
  
  •  The expression of the target protein is inducible by providing T7 RNA polymerase in the host cell as an inducing signal.
  
  
  •  Target gene is initially cloned to host cell that do not contain T7 RNA Polymerase, thus increasing plasmid stability.
  
  
  •  Once stabilized in a non-expression host, the recombinant plasmid is transferred to a expression host having T7 RNA Polymerase gene in the genome.
  
  
  •  Ampicillin and kannamycin resistance genes are available in pET vectors as selection marker.
  
  •  pET28 and pET32 are the most commonly used pET vectors.

1-3.9 Specialized Expression Vectors:

In molecular biology, vectors are generally designed for cloning a foreign gene into a host genome to produce proteins which are not produced by host. But, apart from these applications, different specialized vectors have been constructed to achieve different application in genetic and molecular biology studies and are termed as specialized vectors. Molecular and genetics study of a gene or protein can be aided by specialized vectors. Some of the applications of specialized vectors are discussed below-

1-3.8.1: Promoter Probe Vectors:

Specialized vectors used for identification of efficient promoter region in a DNA segment are termed promoter probe vectors. Promoter-less reported genes (lacZ, GFP etc) are used for construction of promoter probe vectors. The expression of the reporter genes can be monitored and quantified easily using various biochemical or fluorescent techniques. Fusion of DNA fragment containing a promoter region upstream of the reporter gene drives the expression of the reported gene. However, there is no guarantee that the DNA sequence that behaves as promoter in recombinant host can behave in the same way in its native host (Pseudo- promoter). Further characterization is necessary to define a true novel promoter. Some of the widely used promoter probe vectors families are: pOT (eg. pRU1161, pRU1097 etc) and pJP2 (eg. pRU1156, pRU1157 etc). pOT vectors have higher copy number but lower stability as compared to pJP2 vectors.

1-3.9.2 Gene Fusion Vectors:

Fusion of one gene to another gene in order to produce a fusion protein is widely used in molecular biology studies. Fusion proteins are generated by cloning two or more target genes with a reporter gene (His-tag, gfp, rfp, lacZ etc) by using gene fusion vectors. Fusion proteins may provide improved properties like easy isolation and purification of target protein (His-tag), easy monitoring of gene expression level (GFP, RFP, lacZ), intracellular protein localization studies (GFP, RFP, LUC) etc. The target gene is cloned downstream of the promoter region present in the vector. Depending on the requirement, the target protein can be cloned either to the N-terminal or C-terminal of the reporter protein. Different vectors have been commercially available to provide such flexibility in cloning site and reporter gene.