Studies on chemical synthesis of human cystatin A gene and its expression in Escherichia coli.
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.
Kaji
H
H
Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Tokyo.
Kumagai
I
I
Takeda
A
A
Miura
K
K
Samejima
T
T
D001483:Base Sequence; D015853:Cysteine Proteinase Inhibitors; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*
D015853:Cysteine Proteinase Inhibitors; D011480:Protease Inhibitors; D011994:Recombinant Proteins