Expression of the gene encoding cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) in Escherichia coli: export and processing of the apoprotein.
The expression of cytochrome c3 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 c3 (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 c3 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 c3 polypeptide, but fails to insert the haems.
Pollock
W B
WB
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Chemerika
P J
PJ
Forrest
M E
ME
Beatty
J T
JT
Voordouw
G
G
D015153:Blotting, Western; D003574:Cytochrome c Group / Q000096:biosynthesis*; D003901:Desulfovibrio / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D008970:Molecular Weight; D009747:Nutritional Physiological Phenomena; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic
D003574:Cytochrome c Group