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Synthesis, cloning and expression in Escherichia coli of a gene coding for the Met8 → Leu CMTI I — a representative of the squash inhibitors of serine proteinases
Author(s) -
Bolewska Krystyna,
Krowarsch Daniel,
Otlewski Jacek,
Jaroszewski Lukasz,
Bierzynski Andrzej
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)01335-0
Subject(s) - microbiology and biotechnology , fusion protein , chemistry , trypsin inhibitor , cucurbita maxima , gene , biology , gene expression , biochemistry , recombinant dna , trypsin , enzyme , genetics
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 source.