1887

Abstract

The expression of cytochrome from (Hildenborough) was examined in transformed with either of two plasmids, pJ8 and pJ81. The former has an 840 bp insert of DNA, containing the structural gene for cytochrome (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 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 TG2(pJ81) cells, due to the mutation of the signal peptide cleavage site. No insertion of haem into the product could be detected in TG2(pJ8) cells by post-electrophoretic protohaem fluorescence analysis. The sensitivity of the cytochrome synthesized in TG2(pJ8) to digestion by chymotrypsin also indicated that the apoprotein was formed. The results indicate that is capable of synthesizing and exporting the cytochrome polypeptide, but fails to insert the haems.

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1989-08-01
2021-05-17
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