Synthesis of human insulin gene. VIII. Construction of expression vectors for fused proinsulin production in Escherichia coli.
We have constructed two families of plasmids suitable for the cloning of genes and for directing the synthesis of large amounts of fused proteins in Escherichia coli. The plasmids include the E. coli lac promoter and a portion of the coding sequence for beta-galactosidase, which can code for approx. 590 or 450 amino acids. The truncated beta-galactosidase gene ends with a poly-linker region at the 3' end, which can be cleaved by any one of the eight common restriction enzymes and joined to the gene coding for any desired protein. Each family includes three plasmids that enable fusion to be made in all three of the translational reading frames. We have cloned a synthetic human proinsulin gene into these plasmids, and 30% of the total E. coli protein was represented by the 590 amino acid-long truncated beta-galactosidase fused to proinsulin. The yield of proinsulin in this system is more than twice the amount produced by using a 1007 amino acid-long beta-galactosidase gene for fusion.
D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D007328:Insulin / Q000235:genetics*; D007763:Lac Operon; D010957:Plasmids; D011384:Proinsulin / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D007328:Insulin; D011384:Proinsulin; D004262:DNA Restriction Enzymes; D001616:beta-Galactosidase