HIGH-LEVEL SYNTHESIS OF THE 12-KDA HUMAN FK506-BINDING PROTEIN IN ESCHERICHIA-COLI USING TRANSLATIONAL COUPLING

The gene encoding the 12-kDa cytosolic form of human FK506-binding pro tein (hFKBP-12) was isolated from a Jurkat T-cell cDNA library and exp ressed in three different non-fusion systems in Escherichia coli. Expr ession of recombinant hFKBP-12(re-hFKBP-12)from the trc promoter vecto r, pKK233-2, yielded low levels of protein. A second system, which uti lized a modified lac promoter and a stronger ribosome-binding site, sh owed greatly improved expression. A third system, utilizing translatio nal coupling to an upstream segment of kdsB under the control of this modified lac promoter, produced re-hFKPB-12 at a very high level. The re-hFKBP-12 produced via translational coupling was soluble and was sh own to have the authentic N terminus. The level of active re-hFKPB-12 produced from this vector was estimated to be 50% of total soluble pro tein, based on competition with the fusion protein, CKS::re-hFKBP-12, for binding to ascomycin-C22-carboxymethyloxime-alkaline phosphatase.