Efficient production of native, biologically active human cystatin C by Escherichia coli

Abstract

A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the I Ps promoter, an optimized Shine-Dalgamo sequence and the rZ ~I857 repressor. When induced at 42°C such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized.All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated.The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.

Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E. coli as fusion protein and its isolation

“…Only very recently cysteine proteinase inhibitor genes were produced by recombinant DNA methods, namely human cystatin C [33,34], stefin A [35], rat cystatin alpha [36] and stefin B [13,37]. These recombinant inhibitors will facilitate the elucidation of reaction mechanisms, of structure and function relationship and encourage studies on therapeutic effectiveness of these proteins.…”

Cystatin C Properties Crucial for Uptake and Inhibition of Intracellular Target Enzymes

“…In short the pHD313 expression plasmid (17) was used as template for thermal cycling with primers encoding the different mutations. The non-mutated plasmids were digested while the mutated plasmids were purified and electroporated into XL1-Blue electrocompetent cells.…”

“…The bacterial suspensions were used for isolation of plasmids followed by DNA sequencing (AB3010 Genetic Analyzer, BMlabbet, Lund, Sweden). Plasmids which contained correct mutations were electroporated into Escherichia coli MC1061 bacteria for protein expression as described elsewhere (17,18). N-terminally truncated cystatin C was obtained by incubating wild-type cystatin C with leukocyte elastase at the molar ratio 100:1 in 37°C for 4 h.…”

The cysteine protease inhibitors cystatins inhibit herpes simplex virus type 1-induced apoptosis and virus yield in HEp-2 cells

“…The preparation was free of other studied cystatins. Cystatins C and D were produced by expression in Escherichia coli as described previously (Abrahamson et al, 1988;Freije et al, 1993).…”