Bacterial production of latarcin 2a, a potent antimicrobial peptide from spider venom.
Natural venoms are promising sources of candidate therapeutics including antibiotics. A recently described potent antimicrobial peptide latarcin 2a (Ltc 2a) from Lachesana tarabaevi spider venom shows a broad-spectrum antibacterial activity. This peptide consists of 26 amino acid residues and therefore its production using chemical synthesis, although trivial, is costly. We describe an easy approach to Ltc 2a production in Escherichia coli using the conventional fusion partner thioredoxin. Latarcin 2a synthetic gene was cloned into the expression vector pET-32b, which was then used to transform E. coli BL21(DE3) strain. His-tagged fusion purification was achieved using metal-chelate affinity chromatography. Since no methionine residues are present in the latarcin 2a sequence, cyanogen bromide could be effectively utilized to separate the target product from the carrier protein. Reverse-phase HPLC was used as the final step of purification; the final yield was approximately 3 mg/L of bacterial culture. To increase the yields, we attempted incorporation of Ltc 2a tandem repeats into the fusion protein; however, production rates greatly decreased due to enhanced fusion toxicity. Moreover, we probed constructs to produce an Ltc 2a dimer and the Ltc 2a propeptide to study their functional properties. Recombinant peptides were produced at appreciable yields and biological tests to determine their activities were performed. Latarcin 2a is the first linear peptide from spider venom and one of the first membrane-active peptides from venomous animals to be biosynthetically produced.
Shlyapnikov
Yuri M
YM
M.M. Shemyakin & Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya, 16/10, 117997 GSP, Moscow V-437, Russia.
Andreev
Yaroslav A
YA
Kozlov
Sergey A
SA
Vassilevski
Alexander A
AA
Grishin
Eugene V
EV
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D001412:Bacillus subtilis / Q000187:drug effects; D019281:Dimerization; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D013111:Spider Venoms / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D013879:Thioredoxins / Q000235:genetics
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; D013111:Spider Venoms; C531159:latarcin 2a, Lachesana tarabaevi; D013879:Thioredoxins