Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli.
Direct expression of lactoferricin, an antimicrobial peptide, is lethal to Escherichia coli. For the efficient production of lactoferricin in E. coli, we developed an expression system in which the gene for the lysine- and arginine-rich cationic lactoferricin was fused to an anionic peptide gene to neutralize the basic property of lactoferricin, and successfully overexpressed the concatemeric fusion gene in E. coli. The lactoferricin gene was linked to a modified magainin intervening sequence gene by a recombinational polymerase chain reaction, thus producing an acidic peptide-lactoferricin fusion gene. The monomeric acidic peptide-lactoferricin fusion gene was multimerized and expressed in E. coli BL21(DE3) upon induction with isopropyl-beta-D-thiogalactopyranoside. The expression levels of the fusion peptide reached the maximum at the tetramer, while further increases in the copy number of the fusion gene substantially reduced the peptide expression level. The fusion peptides were isolated and cleaved to generate the separate lactoferricin and acidic peptide. About 60 mg of pure recombinant lactoferricin was obtained from 1 L of E. coli culture. The purified recombinant lactoferricin was found to have a molecular weight similar to that of chemically synthesized lactoferricin. The recombinant lactoferricin showed antimicrobial activity and disrupted bacterial membrane permeability, as the native lactoferricin peptide does.
Kim
Ha-Kun
HK
Department of Genetic Engineering, PaiChai University, 439-6, Doma-dong, Seo-ku, Daejon, 302-735, South Korea.
Chun
Dae-Sik
DS
Kim
Joon-Sik
JS
Yun
Cheol-Ho
CH
Lee
Ju-Hoon
JH
Hong
Soon-Kwang
SK
Kang
Dae-Kyung
DK
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D007781:Lactoferrin / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D010455:Peptides; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D007781:Lactoferrin