Recombinant production and purification of novel antisense antimicrobial peptide in Escherichia coli.
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.
Haught
C
C
School of Chemical Engineering and Materials Science, University of Oklahoma, Norman, Oklahoma 73019-0628, USA.
Davis
G D
GD
Subramanian
R
R
Jackson
K W
KW
Harrison
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification*; D001709:Biotechnology; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D000900:Anti-Bacterial Agents; D010455:Peptides; D011993:Recombinant Fusion Proteins