Expression of the Gene Encoding Cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) in Escherichia coli: Export and Processing of the Apoprotein
Autor: | M. E. Forrest, P. J. Chemerika, W. B. R. Pollock, J. T. Beatty, G. Voordouw |
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Rok vydání: | 1989 |
Předmět: |
Cytochrome
Blotting Western Cytochrome c Group medicine.disease_cause Microbiology Transformation Genetic Escherichia coli medicine Nutritional Physiological Phenomena Site-directed mutagenesis Desulfovibrio vulgaris Gel electrophoresis Signal peptidase biology Cytochrome c Structural gene Gene Expression Regulation Bacterial biology.organism_classification Molecular biology Molecular Weight Biochemistry biology.protein Desulfovibrio Electrophoresis Polyacrylamide Gel Plasmids |
Zdroj: | Microbiology. 135:2319-2328 |
ISSN: | 1465-2080 1350-0872 |
Popis: | SUMMARY: The expression of cytochrome c 3 from Desulfovibrio vulgaris (Hildenborough) was examined in Escherichia coli transformed with either of two plasmids, pJ8 and pJ81. The former has an 840 bp insert of D. vulgaris DNA, containing the structural gene for cytochrome c 3 (387 bp) and its promoter region. Plasmid pJ81 was generated from pJ8 by deoxyoligonucleotide-directed mutagenesis to direct the synthesis of a protein with an altered signal peptidase cleavage site [Ala(-- 1).Asp(-- 1)]. Synthesis of the 14 kDa precursor, which was partly processed to the 12 kDa mature protein, was observed in cells of E. coli TG2(pJ8) by SDS gel electrophoresis and Western blotting. Analysis of spheroplasts revealed that the processed polypeptide was present in the periplasm while the precursor was found only in the membrane/cytoplasmic fraction. No processing was observed in E. coli TG2(pJ81) cells, due to the mutation of the signal peptide cleavage site. No insertion of haem into the E. coli product could be detected in E. coli TG2(pJ8) cells by post-electrophoretic protohaem fluorescence analysis. The sensitivity of the cytochrome c 3 synthesized in E. coli TG2(pJ8) to digestion by chymotrypsin also indicated that the apoprotein was formed. The results indicate that E. coli is capable of synthesizing and exporting the cytochrome c 3 polypeptide, but fails to insert the haems. |
Databáze: | OpenAIRE |
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