High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage.
Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation, and decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners. To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR residues present at the C terminus of histonin, furin cleavage of the multimeric histonin expressed produces an intact, natural histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 l of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host.
Kim
Jung Min
JM
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, South Korea.
Jang
Su A
SA
Yu
Byung Jo
BJ
Sung
Bong Hyun
BH
Cho
Ju Hyun
JH
Kim
Sun Chang
SC
D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D000935:Antifungal Agents / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005658:Fungi / Q000187:drug effects; D045683:Furin / Q000378:metabolism*; D015870:Gene Expression; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; D045683:Furin