Biologically active and C-amidated hinnavinII-38-Asn produced from a Trx fusion construct in Escherichia coli.
The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn (HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15-20% of the total cell proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography, and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus.
Kang
Chang Soo
CS
Department of Biological Science and the Research Institute for Basic Sciences, Hoseo University, Asan, 336-795, Republic of Korea.
Son
Seung-Yeol
SY
Bang
In Seok
IS
D000577:Amides / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001216:Asparagine / Q000737:chemistry* / Q000378:metabolism; D001419:Bacteria / Q000187:drug effects; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D000577:Amides; D000900:Anti-Bacterial Agents; D029968:Escherichia coli Proteins; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D001216:Asparagine