Cloning and expression in Escherichia coli of DNA encoding a 60 kDa stress protein of Mycobacterium paratuberculosis, the causative agent of Johne's disease
| Autor: | Raymond Bujdoso, Angela Colston, Ian McConnell |
|---|---|
| Rok vydání: | 1994 |
| Předmět: |
DNA
Bacterial Molecular Sequence Data Restriction Mapping Gene Expression Paratuberculosis Biology Polymerase Chain Reaction Microbiology law.invention HSPA4 Bacterial Proteins law HSPA2 Escherichia coli medicine Animals Amino Acid Sequence Cloning Molecular Peptide sequence Heat-Shock Proteins DNA Primers Expression vector HSPA14 Base Sequence medicine.disease Fusion protein Molecular biology Recombinant Proteins Molecular Weight Mycobacterium avium subsp. paratuberculosis Biochemistry Recombinant DNA Plasmids |
| Zdroj: | Microbiology. 140:3329-3336 |
| ISSN: | 1465-2080 1350-0872 |
| Popis: | Polymerase chain reaction (PCR) was used to generate DNA encoding a 60 kDa stress protein of Mycobacterium paratuberculosis using primers complementary to sequences at the 5' and 3' ends of 60 kDa stress protein genes (encoding the '65 kDa antigens') of M. leprae and M. tuberculosis. The predicted PCR product of 1.8 kb contained the entire coding sequence of an M. paratuberculosis 60 kDa stress protein, with non-coding regions of 124 bp and 1 bp at the 5' and 3' ends, respectively. DNA encoding the entire ORF for the 60 kDa stress protein, as well as thrombin and Factor Xa proteolytic cleavage sites, was ligated into the bacterial expression vector pGEX-2T and used to transform Escherichia coli strain JM83. Transformed bacteria, induced by IPTG, expressed an 85 kDa fusion protein comprising glutathione S-transferase (GST) and M. paratuberculosis 60 kDa stress protein. This fusion protein was purified by adsorption to glutathione-agarose beads and shown to cross-react in Western blot analysis with an anti-mycobacterial 60 kDa stress protein monoclonal antibody. Recombinant M. paratuberculosis 60 kDa stress protein was liberated from GST by proteolytic cleavage with either thrombin or Factor Xa enzyme. Authenticity of liberated recombinant stress protein was confirmed by N-terminal amino acid sequencing. |
| Databáze: | OpenAIRE |
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