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.
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 beta-trypsin and human cathepsin G are the same, within experimental error, as for CMTI I isolated from a natural source.
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
K
K
Bolewska
D
D
Krowarsch
J
J
Otlewski
L
L
Jaroszewski
A
A
Bierzynski
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D018744:DNA, Plant; D004926:Escherichia coli; D006801:Humans; D007930:Leucine / Q000235:genetics; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014675:Vegetables / Q000737:chemistry* / Q000235:genetics
C081870:CMTI protein, Cucurbita maxima; D018744:DNA, Plant; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D008715:Methionine; D007930:Leucine