Cloning and Expression Vectors

All Vectors used for propagation of DNA insers in a suitable host are called cloning vectors. But when a vector is designed for the expression of production of the protein sepcified by the DNA insert, itis termed as expression vector. As a rule such vectors contian atleast the regulatory sequences, promoters, operators ribosomal binding sites ets, having optimum function in the chosen host. It is desirable that all cloning vectors have relaed replication control so that they can produce multiple copies per host cell.
When an eukaryotic gene to be expressed i a prokaryote, the eukaryotic coding sequence has to be placed after prokaryotic promoter and ribosome buildin site since the regulatory sequences of eukaryotic are not recognised in prokaryotes in addition, eukaryotic genes as a rule, contain introns presend 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 issolated 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 (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 expressions insulin fat growth hormone, structural protein VPI of foot and mouth disease virus, human growth hormones etc. Some examples of the second approach producing unique proteins are rabbit B globin, small antigen of SV 40, human fibroblast interferon, human human IGF-1 protein. It may be pointed out that the undesired amino acids encoded by the vector sequence in case 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 from precusor proteins to obtain active mature protein molecules. Various strategies are being rapidly devised to effectively overcome these problems.