Optimization of the β- galactosidase polypeptide Length for the overproduction of fusion proteins in E. coli.

Abstract

Plasmid vectors for cloning and directing the synthesis of large amounts of fusion proteins in E. coll were constructed. These plasmids carried tac promoter, the coding sequence of amino terminal portion of B-galactosidase, and the polylinker region at the 3z-end of truncated lacZ. When E. coli MC1061 cells were transformed with these plasmids, 25-60% of the total cellular proteins were represented by the lacZ polypeptides. The overproduced proteins formed inclusion bodies inside the cells. When the amounts of recombinant proteins produced by these plasmids were examined, the higher yield was observed with those of shorter lacZ polypeptides. The length of recombinant polypeptide with the maximum yield (60%) was about 280 amino acids long. However, when the length of 13-galactosidase decreased to 200 residues as in pCT30 vector, the yield of recombinant proteins decreased dramatically. When the length of polypeptide increased again to 260 residues by fusing pre-S2 sequence of hepatitis B virus to lacZ sequence in pCT30, the yield of fusion polypeptides increased to over 50% of total cellular proteins.