Expression of recombinant hybrid peptide hinnavin II/alpha-melanocyte-stimulating hormone in Escherichia coli: purification and characterization.
The increasing problem of antibiotic resistance among pathogenic bacteria requires novel strategies for the construction of multiple, joined genes of antimicrobial agents. The strategy used in this study involved synthesis of a cDNA-encoding hinnavin II/alpha-melanocyte-stimulating hormone (hin/MSH) hybrid peptide, which was cloned into the pET32a (+) vector to allow expression of the hybrid peptide as a fusion protein in Escherichia coli BL21 (DE3). The resulting expression of fusion protein Trx-hin/MSH could reach up to 20% of the total cell proteins. More than 50% of the target protein was in a soluble form. The target fusion protein from the soluble fraction, Trx-hin/MSH, was easily purified by Ni(2+)-chelating chromatography. Then, enterokinase cleavage effectively cleaved the Trx-hin/MSH to release the recombinant hin/MSH (rhin/MSH) hybrid peptide. After removing the contaminants, we purified the recombinant hybrid peptide to homogeneity by reversed-phase FPLC and obtained 210 mg of pure, active rhin/MSH from 800 ml of culture medium. Antimicrobial activity assay demonstrated that rhin/MSH had a broader spectrum of activity than did the parental hinnavin II or MSH against fungi and Gram-positive and Gram-negative bacteria. These results suggest an efficient method for producing high-level expression of various kinds of antimicrobial peptides that are toxic to the host, a reliable and simple method for producing different hybrid peptides for biological studies.
Bang
Son Kwon
SK
Department of Biological Science, Hoseo University, Asan, Republic of Korea.
Kang
Chang Soo
CS
Han
Man-Deuk
MD
Bang
In Seok
IS
D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000521:alpha-MSH / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; C550329:hinnavin II, Artogeia rapae; D000521:alpha-MSH