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Home » Biology Homework Help » Biotechnology » Cloning and Expressing Vectors
Cloning and Expressing Vectors
All vectors used for propagation of DNA inserts in a suitable host are called cloning vectors. But when a vector is designated for the expression of i.e. production of the protein specified by, the DNA insert, it is termed as expression vector. As a rule, such vectors, contain at least the regulatory sequences, i.e. promoters, operators, ribosomal binding sites etc, having optimum function in the chosen host. It is desirable that all cloning vectors have relaxed replication control so that they can produce multiple copies per host cell.

When an eukaryotic gene to be expressed in prokaryote, the eukaryotic coding sequence has to be placed after prokaryotic promoter and ribosome building sites since the regulatory sequences of eukaryotic are not recognized in prokaryotes. In addition, eukaryotic genes, as a rule, contain introns (noncoding regions) present within their coding regions. These introns must be removed to enable the proper expression of eukaryotic genes since prokaryotes lack the machinery needed for their removal from the RNA transcripts. When eukaryotic genes are isolated as cDNA they are intron-free and, hence suitable for expression in prokaryotes.

Several strategies have been attempted for the construction of expression vectors using regulatory sequences of the appropriate hosts. These approaches may be grouped into the following two broad categories.

1. Construction of vectors allowing the synthesis of fusion proteins comprising amino acids coded by a sequence in the vector and those encoded by the DNA insert (translational fusion).

2. Development of vectors permitting the synthesis of pure proteins encoded exclusively by the DNA inserts (transcriptional fusion).

Examples of the first strategy producing fusion proteins are: the expression of rat insulin, rat growth hormone, structural protein VPI of foot and mouth disease virus, human growth hormone etc. Some examples of the second approach producing unique proteins are: rabbit – globin, small t-antigen of SV 40, human fibroblast interferon, human IGF-I protein. It may be pointed out that the undesired amino acids encoded by the vector sequence in cases of translational fusion must be removed from the fusion proteins by a suitable chemical cleavage.

Several other problems are faced when eukaryotic genes are expressed in a prokaryotic system, e.g. removal of signal sequences form precursor proteins to obtain active mature protein molecules. Various strategies are being rapidly devised to effectively overcome these problems.

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