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Recombinant production and purification of novel antisense antimicrobial peptide in Escherichia coli
Author(s) -
Haught Chris,
Davis Gregory D.,
Subramanian Rajesh,
Jackson Ken W.,
Harrison Roger G.
Publication year - 1998
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19980105)57:1<55::aid-bit7>3.0.co;2-u
Subject(s) - recombinant dna , antimicrobial , peptide , microbiology and biotechnology , biochemistry , chemistry , biology , gene
A fusion protein was genetically engineered that contains an antimicrobial peptide, designated P2, at its carboxy terminus and bovine prochymosin at its amino terminus. Bovine prochymosin was chosen as the fusion partner because of its complete insolubility in Escherichia coli, a property utilized to protect the cells from the toxic effects of the antimicrobial peptide. This fusion protein was purified by centrifugation as an insoluble inclusion body. A methionine linker between prochymosin and the P2 peptide enabled P2 to be released by digestion with cyanogen bromide. Cation exchange HPLC followed by reversed‐phase HPLC were used to purify the P2 peptide. The recombinant P2 peptide's molecular mass was confirmed by mass spectrometry to within 0.1% of the theoretical value (2480.9 Da), and the antimicrobial activity of the purified recombinant P2 against E. coli D31 was determined to be identical to that of the chemically synthesized peptide (minimal inhibitory concentration of 5 mg/mL). Although the yield of the fusion protein after expression by the cells was high (16% of the total cell protein), the percentage recovery of the P2 peptide in the inclusion bodies was relatively low, which appears to be due to losses in the cyanogen bromide digestion step. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 55–61, 1998.