Production and purification of a cecropin family antibacterial peptide, hinnavin Ⅱ, in Escherichia coli

• Published in: Biotechnology and bioprocess engineering Vol. 13; no. 3; pp. 377 - 382
• Main Authors: Kang, C.S. (Hoseo University, Asan, Republic of Korea), Park, C.W. (Hoseo University, Asan, Republic of Korea), Bang, I.S. (Hoseo University, Asan, Republic of Korea), E-mail: isbang@hoseo.edu
• Format: Journal Article
• Language: English
• Published: Heidelberg The Korean Society for Biotechnology and Bioengineering 01.06.2008; Springer Nature B.V; 한국생물공학회
• Subjects: antibacterial peptide; Artogeia rapae; Bacillus megaterium; Biotechnology; Brassica; Chemistry; Chemistry and Materials Science; E coli; Enterobacter cloacae; ESCHERICHIA COLI; fusion expression; Industrial and Production Engineering; Peptides; recombinant production; Staphylococcus aureus; 생물공학; antibacterial peptide; fusion expression; hinnavin II; recombinant production
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-008-0044-1

The antibacterial peptide hinnavin Ⅱ, isolated from the cabbage butterfly Artogeia rapae, is synthesized with an amidated lysine 37 residue at C-terminus. Glycine-extended native hinnavin Ⅱ (hinnavin Ⅱ-38-Gly, hin Ⅱ) gene with 114 bp coding region was cloned in the expression vector pET-32a (+) to construct a fusion expression plasmid and transformed into Escherichia coli BL21 (DE3) pLysS. The recombinant fusion protein Trx-hin Ⅱ was expressed in soluble form, purified successfully by Ni²+-chelating chromatography, and cleaved by enterokinase to release recombinant hin Ⅱ (rhin Ⅱ). Purification of the rhin Ⅱ was achieved by reversed-phase FPLC, and 2.45 mg pure active rhin Ⅱ was obtained from 800 mL E. coli culture. The molecular mass of the rhin Ⅱ determined by MALDI-TOF mass spectrometry is consistent with the theoretical molecular mass of 4,195.0 Da. The purified rhin Ⅱ showed antimicrobial activities against tested E. coli K12, E. coli BL21 (DE3), Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus. The application of this expression/purification approach represents a fast and efficient method to prepare milligram quantities of hinnavin Ⅱ in its biologically active form.