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 protein (hFKBP-12) was isolated from a Jurkat T-cell cDNA library and expressed in three different non-fusion systems in Escherichia coli. Expression of recombinant hFKBP-12 (re-hFKBP-12) from the trc promoter vector, pKK233-2, yielded low levels of protein. A second system, which utilized a modified lac promoter and a stronger ribosome-binding site, showed greatly improved expression. A third system, utilizing translational 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 shown to have the authentic N terminus. The level of active re-hFKBP-12 produced from this vector was estimated to be 50% of total soluble protein, based on competition with the fusion protein, CKS::re-hFKBP-12, for binding to ascomycin-C22-carboxymethyloxime-alkaline phosphatase.
Pilot-Matias
T J
TJ
Abbott Laboratories, Abbott Park, IL 60064.
Pratt
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D000469:Alkaline Phosphatase / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005796:Genes; D006801:Humans; D007763:Lac Operon; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D016559:Tacrolimus / Q000031:analogs & derivatives; D022021:Tacrolimus Binding Proteins
D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; C058028:immunomycin; D009713:Nucleotidyltransferases; C028984:3-deoxy-manno-octulosonate cytidyltransferase; D000469:Alkaline Phosphatase; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus