Processing and secretion by Escherichia coli of a recombinant form of the immunogenic protein MPB70 of Mycobacterium bovis.
The gene encoding an immunodominant secreted antigen, MPB70, of Mycobacterium bovis was cloned into the plasmid vector pBluescript II KS+ along with its native ribosome-binding site. In this construct translation of the protein in Escherichia coli was from the native AUG initiation codon and was directed by the mycobacterial ribosome-binding site. Two different molecular mass forms (26 kDa and 22 kDa) of MPB70 were observed in whole-cell pellets of recombinant E. coli. The difference in size indicates cleavage of the signal peptide of MPB70 by an endopeptidase of E. coli. MPB70 was secreted into the periplasm of recombinant E. coli, where the 22 kDa form of the protein was predominant. The culture filtrate contained only the 22 kDa form of the protein, which was soluble. The passage of MPB70 from the periplasm into the growth medium was found to be due, at least in part, to non-specific leakage of periplasmic proteins across the outer membrane associated with the expression of recombinant MPB70.
Hewinson
R G
RG
Molecular Genetics Unit, Central Veterinary Laboratory, New Haw, Weybridge, Surrey, UK.
Russell
W P
WP
D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000235:genetics* / Q000276:immunology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C060216:MPB70 protein, Mycobacterium bovis