Synthesis, cloning and expression inEscherichia coliof a gene coding for the Met8 → Leu CMTI I - a representative of the squash inhibitors of serine proteinases
- 18 December 1995
- journal article
- Published by Wiley in FEBS Letters
- Vol. 377 (2) , 172-174
- https://doi.org/10.1016/0014-5793(95)01335-0
Abstract
A chemically synthesized gene coding for a Cucurbita maxima trypsin inhibitor modified at position P′3 (Met8→ Leu CMTI I), i.e. at the third position downstream of the reactive site bond (Arg5-Ile), was cloned into a derivative of the plasmid pAED4 that utilizes a T7 expression system. The gene was expressed in Escherichia coli as a fusion protein that accumulates in inclusion bodies. After reduction and CNBr cleavage of the fusion protein followed by oxidative refolding and reverse-phase HPLC, about 5 mg of pure protein was obtained per 1 of cell culture. Association constants of recombinant Leu-8-CMTI I with bovine β-trypsin and human cathepsin G are the same, within experimental error, as for CMTI I isolated from a natural sourceKeywords
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