Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis
| Autor: | Jan Kok, J M van Dijl, Gerhardus Venema, J. M. B. M. Van Der Vossen |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1985 |
| Předmět: |
DNA
Bacterial DNA Recombinant Bacillus subtilis Deoxyribonuclease HindIII HindIII Molecular cloning medicine.disease_cause Applied Microbiology and Biotechnology Plasmid Endopeptidases medicine Animals Cloning Molecular Escherichia coli Electrophoresis Agar Gel Ecology biology Lactococcus lactis Nucleic Acid Hybridization Streptococcus DNA Restriction Enzymes biology.organism_classification Molecular biology Restriction enzyme Milk Phenotype biology.protein Transformation Bacterial Immunoelectrophoresis Two-Dimensional Food Science Biotechnology Research Article Plasmids |
| Popis: | Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of Streptococcus cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed, the entire plasmid was subcloned in Escherichia coli with plasmids pBR329 and pACYC184. A 4.3-Md HindIII fragment could not be cloned in an uninterrupted way in E. coli but could be cloned in two parts. Both fragments showed homology with the 9-Md proteinase plasmid of S. cremoris HP. The 4.3-Md HindIII fragment was successfully cloned in Bacillus subtilis on plasmid pGKV2 (3.1 Md). Crossed immunoelectrophoresis of extracts of B. subtilis carrying the recombinant plasmid (pGKV500; 7.4 Md) showed that the fragment specifies two proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient Streptococcus lactis strain via protoplast transformation. Both proteins were also present in cell-free extracts of S. lactis(pGKV500). In S. lactis, pGKV500 enables the cells to grow normally in milk with rapid acid production, indicating that the 4.3-Md HindIII fragment of plasmid pWV05 specifies the proteolytic activity of S. cremoris Wg2. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|