Expression of a functional rat salivary cystatin S polypeptide in Escherichia coli.
Cystatin S is a cysteine proteinase inhibitor that is transiently expressed during rat submandibular gland development and can be induced by isoproterenol in the adult. A cDNA for rat cystatin S which included the entire coding sequence of the secreted cystatin was cloned. A coding region of the cystatin gene was amplified by polymerase chain reaction and cloned into the pGEX-2T expression vector. The chimeric plasmid was transformed into Escherichia coli, and protein expression was induced by isopropyl-beta-D-thiogalactopyranoside. The expressed protein was purified from insoluble inclusion bodies after solubilization with urea and fast protein liquid chromatography on a MonoQ column. The purified recombinant cystatin reacted with antibodies to cystatin S purified from rat submandibular glands and showed an amino-terminal amino acid sequence identical to that of rat cystatin S. The recombinant protein exhibited papain inhibition activity comparable to natural cystatin. This was a successful expression and purification of a functionally and immunologically reactive recombinant cystatin from E. coli, an approach which will be used later towards generating recombinant variants to study the binding and functional domains of this cysteine protease inhibitor.
Sharma
A
A
Department of Oral Biology, State University of New York at Buffalo, USA.
O'Connell
B C
BC
Tabak
L A
LA
Bedi
G S
GS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001586:Benzoylarginine Nitroanilide; D001681:Biological Assay; D003001:Cloning, Molecular; D015891:Cystatins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015853:Cysteine Proteinase Inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D055333:Salivary Cystatins; D012471:Salivary Proteins and Peptides / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013363:Submandibular Gland / Q000378:metabolism
C525174:Cyss protein, rat; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D011994:Recombinant Proteins; D055333:Salivary Cystatins; D012471:Salivary Proteins and Peptides; D001586:Benzoylarginine Nitroanilide