Production of rat salivary cystatin S variant polypeptides in Escherichia coli.
Cystatins are protein inhibitors of papain and related cysteine proteinases. A series of continuous synthetic peptides corresponding to the entire sequence of rat salivary cystatin was used to localize the binding domains of the cystatin to papain. Several synthetic peptides, one from the aminoterminal sequence (peptide 1-24) and others from the carboxylterminal (peptides 66-79, 66-90, 79-90, 79-114), showed binding to papain, but none of the peptides showed inhibition of papain activity. Three recombinant rat salivary cystatin variants (N-terminal truncated protein lacking amino acid residues 1-9; variant 49-53, in which amino acid residues QVVAG of rat salivary cystatin had been replaced with amino acid residues LVL in mutant protein; and variant 65-78, in which amino acid residues 65-78 had been replaced with amino acids PG in mutant protein) were produced using the Escherichia coli expression system pGex-4T. To generate N-terminal truncated protein the desired coding region of the cystatin gene was amplified by polymerase chain reaction (PCR). To produce the variants 49-53 and 65-78, a PCR-based approach of gene splicing by overlap extension was used. Recombinant cystatin proteins were produced as insoluble inclusion bodies as fusion proteins with a glutathione S-transferase (GST) carrier. After solubilization with urea the GST carrier was cleaved from the fusion protein with thrombin and cystatin variants purified by fast liquid chromatography on a MonoQ column. The purified proteins reacted with antibodies to rat salivary cystatin. The N-terminal truncated and variant 49-53 exhibited very little inhibitory activity towards papain, whereas variant 65-78 exhibited papain-inhibitory activity similar to the full-length recombinant cystatin.
Bedi
G S
GS
Department of Microbiology and Immunology, Medical College of Pennsylvania, Philadelphia, USA. bedi@auhs.edu
Zhou
T
T
Bedi
S K
SK
D017398:Alternative Splicing; D000596:Amino Acids / Q000235:genetics; D000818:Animals; D000906:Antibodies; D002352:Carrier Proteins / Q000235:genetics; D002853:Chromatography, Liquid; D015891:Cystatins / Q000235:genetics*; D015853:Cysteine Proteinase Inhibitors / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D002479:Inclusion Bodies / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010206:Papain / Q000037:antagonists & inhibitors; D010455:Peptides / Q000235:genetics; D016133:Polymerase Chain Reaction; D051381:Rats; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D055333:Salivary Cystatins; D012471:Salivary Proteins and Peptides / Q000235:genetics*; D012995:Solubility; D013917:Thrombin / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D000596:Amino Acids; D000906:Antibodies; D002352:Carrier Proteins; C525174:Cyss protein, rat; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D055333:Salivary Cystatins; D012471:Salivary Proteins and Peptides; D014508:Urea; D005982:Glutathione Transferase; D013917:Thrombin; D010206:Papain