Studies on Chemical Synthesis of Human Cystatin A Gene and Its Expression in Escherichia coli

Abstract

A synthetic gene containing the coding sequence for the human cysteine proteinase inhibitor, cystatin A, was obtained by enzymatic assembly of 20 oligodeoxyribonucleotides which had been chemically synthesized by the solid phase phosphoramidite method. It was cloned into an Escherichia coli plasmid. The expression plasmid for cystatin A was constructed by introducing the synthetic gene downstream of the tac promoter of an E. coli plasmid which is a derivative of pKK223-3 with high copy number. The gene was expressed in E. coli JM109 without IPTG-induction. The expression of cystatin A was detected by SDS-polyacrylamide gel electrophoresis of the E. coli JM109 lysate, followed by immunoblotting using rabbit antiserum raised with human epidermal cystatin A and alkaline phosphatase-conjugated goat anti-rabbit IgG. The result showed that the molecular weight of the expression product is identical with that of the authentic protein and the antigenic properties are also the same. Furthermore, the expression product purified with a CM-papain Sepharose affinity column and FPLC system with a Mono-Q column showed the same inhibitory activity for various cysteine proteinases. Also, purified recombinant cystatin A was found to have identical amino acid composition, NH2-terminal amino acid sequence, and peptide-map on reverse phase HPLC with those of the authentic inhibitor.

Quail cystatin: Isolation and characterisation of a new member of the cystatin family and its hypothetical interaction with cathepsin B

“…Surprisingly, a significant difference was found in the inhibition of human cathepsin B by quail cystatin when compared with other cystatins. Quail cystatin seems to be the strongest cathepsin B inhibitor ( K i =48 pM) among the cystatins if compared with Stefin A (4–11.2 nM) [42–44], Stefin B (16–130 nM) [45, 46], bovine parotid gland cystatin C (4.4 nM) [40]or human cystatin C (0.17–0.26 nM) [22, 48], having in mind that comparison of K i values obtained under different conditions is difficult.…”

Cystatins — Inhibitors of Cysteine Proteinases

“…The family 2 cystatins that have been studied include (1) a chemically synthesized human cystatin C gene (Strauss et al, 1988); (2) an authentic cystatin C cDNA Dalb0ge et al, 1989); and (3) a synthetic gene-encoding chicken egg white cystatin (Auerswald et al, 1989). The family 1 cystatins include a chemically synthesized human cystatin A gene (Kaji et al, 1989 and1990) and a synthetic gene-encoding rat cystatin a (Katunuma et al, 1988). In all cases, these full-length recombinant cystatins purified from E. coli were similar to the natural ones with respect to their cysteine proteinase inhibitor activity.…”

Characterization by spectroscopic, kinetic and equilibrium methods of the interaction between recombinant human cystatin A (stefin A) and cysteine proteinases

“…Compared with chicken cystatin and cystatins B and C, cystatin A has been relatively little characterized. The human inhibitor has been isolated from polymorphonuclear granulocytes and liver [10,11] and expressed in Escherichia coli [12][13][14][15]. Homologous inhibitors have also been purified from rat and bovine skin [16,17].…”

“…The X-ray structure of cystatin A has not been determined, but a recent analysis by NMR shows a similar overall structure to cystatin B [18]. Published equilibrium constants for the binding of the human inhibitor to cysteine proteinases are markedly discrepant for several proteinases, and rate constants have been reported only for the binding to two enzymes [11][12][13]19,20]. Results from site-directed mutagenesis of residues in the QWAG region have been taken to indicate an insignificant role for this region in proteinase binding [13], in contrast with similar studies with chicken cystatin [21].…”