Intracellular expression of BPTI fusion proteins and single column cleavage/affinity purification by chymotrypsin.
The novel and efficient expression system described here produces formerly poorly expressed, proteolytically unstable mutants of bovine pancreatic trypsin inhibitor (BPTI). A new, single column method for the cleavage of a recombinant fusion to BPTI and affinity purification of the BPTI moiety by immobilized chymotrypsin is an integral part of the system. Wild-type and mutant BPTI molecules are expressed in Escherichia coli as fusion proteins forming intracellular inclusion bodies. Transcription initiation is under the control of the E. coli trp promoter. The expressed protein is tripartite fusion comprising (i) a portion of the TrpLE leader peptide, (ii) a synthetic IgG binding domain derived from protein A and (iii) the BPTI variant. Solubilization of the inclusion bodies and refolding of the fusion proteins in a thiol-disulfide shuffling system yields correctly folded inhibitor molecules. In the single column purification and cleavage procedure, immobilized chymotrypsin cleaves the refolded fusion protein and releases affinity purified active BPTI mutants with correct N-termini. Mutant BPTI molecules which do not fold into active inhibitors are also stably expressed in inclusion bodies but cannot be purified by this method. Unlike previously described secretion systems for the production of BPTI, expression levels in this system appear to be independent of both the mutation in the BPTI gene and the activity of the expressed protein. Mutants poorly expressed in secretion systems can now be produced in sufficient quantities for protein folding studies and structural analysis using X-ray crystallography and NMR spectroscopy.
Altman
J D
JD
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Henner
D
D
Nilsson
B
B
Anderson
S
S
Kuntz
I D
ID
D000595:Amino Acid Sequence; D007611:Aprotinin / Q000235:genetics*; D001483:Base Sequence; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006868:Hydrolysis; D002479:Inclusion Bodies / Q000382:microbiology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D007611:Aprotinin; D002918:Chymotrypsin