A simple method for the purification of an antimicrobial peptide in recombinant Escherichia coli.
A magainin derivative, designated MSI-344, was produced in Escherichia coli as fusion protein, by utilizing a truncated amidophsphoribosyltransferase of E. coli as a fusion partner. Bacterial cells transformed with the gene encoding the fusion protein were grown to a high cell density and induced with isopropyl-1-thio-b-D-galatoside (IPTG) to initiate product expression. The fusion protein was accumulated into cytoplasmic inclusion body and recombinant MSI-344 was released from the fusion partner by hydroxylamine treatment. Following cleavage of the fusion protein with hydroxylamine, the released MSI-344 was purified to homogeneity by cationic exchange chromatography. The final purity was at least 95% by reversed-phase high performance liquid chromatography (RP-HPLC). Purified recombinant MSI-344 was found to be indistinguishable from the synthetic peptide determined by amino acid sequences and antimicrobial activity assay.
Hwang
S W
SW
Department of Chemical Engineering, Kongju National University, 182 Shinkwan-Donk, Kongju 314-701, Chungnam, South Korea.
Lee
J H
JH
Park
H B
HB
Pyo
S H
SH
So
J E
JE
Lee
H S
HS
Hong
S S
SS
Kim
J H
JH
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents / Q000302:isolation & purification* / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004926:Escherichia coli / Q000187:drug effects; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects
D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; C445401:MSI-344 peptide; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins