1. Cloning and expression of a clamworm antimicrobial peptide perinerin in Pichia pastoris
Qingfeng Zhou, Mingyue Li, Tao Xi Curr Microbiol. 2009 Apr;58(4):384-8. doi: 10.1007/s00284-009-9372-4. Epub 2009 Feb 7.
Perinerin, an antimicrobial peptide (GenBank No. P84117), was isolated and characterized from Asian marine clamworms, Perinereis aibuhitensis Grube. This peptide has effects against both gram-positive and gram-negative bacteria in vitro, especially on Pseudemonas aeruginosa. In our study, bioactive perinerin was expressed in Pichia pastoris, and characterized its physicochemical properties. The expressed sample was firstly analyzed by Tricine-SDS-PAGE, after then the recombinant proteins were purified by 2 kD MW cut-off (MWCO) ultrafiltration membrane, and finally purified by 10 kD MWCO ultrafiltration membrane and CM 52-ion exchange chromatography. About 6% protein was obtained by this so called three-purification method. Our results showed that Pichia pastoris was a suitable system for secreting perinerin. Bioactivity assay proved that the recombinant perinerin had antimicrobial effects.
2. High-level production of a novel antimicrobial peptide perinerin in Escherichia coli by fusion expression
Qing-Feng Zhou, Xue-Gang Luo, Liang Ye, Tao Xi Curr Microbiol. 2007 May;54(5):366-70. doi: 10.1007/s00284-006-0466-y. Epub 2007 May 4.
Perinerin is a small antimicrobial peptide (AMP) isolated from an Asian marine clamworm, Perinereis aibuhitensis Grube. It shows marked activity in vitro against both Gram-negative and Gram-positive bacteria. To obtain it in large amounts, the coding sequence of perinerin was cloned into pET32a(+) vector and expression as a Trx fusion protein in Escherichia coli. The soluble fusion protein collected from the supernatant of the cell lyste was separated by Ni(2+)-chelating chromatography. The purified protein was then cleaved by Factor Xa protease to release mature perinerin. Final purification was achieved by ion-exchange chromatography. Recombinant perinerin exhibited a similar antimicrobial activity to the native perinerin. These works might provide a significant foundation for the following research on the action of mechanism of marine AMPs.
3. Perinerin, a novel antimicrobial peptide purified from the clamworm Perinereis aibuhitensis grube and its partial characterization
Weidong Pan, Xianghui Liu, Feng Ge, Jie Han, Tao Zheng J Biochem. 2004 Mar;135(3):297-304. doi: 10.1093/jb/mvh036.
A novel antimicrobial peptide was isolated and partially characterized from the homogenate of an Asian marine clamworm, Perinereis aibuhitensis Grube. This novel peptide, named Perinerin, was purified to homogeneity by heparin-affinity column and reverse-phase HPLC, and biologically tested with a MTS-PMS colorimetric assay. Perinerin consists of 51 amino acid residues and structurally appears to be highly basic and hydrophobic. It shows marked activity in vitro against both Gram-negative and Gram-positive bacteria and fungi, which indicates a bactericidal effect as well. Perinerin appears to be constitutively present and its sequence is novel among all other known antimicrobial peptides. These results suggest that Perinerin has the potential to serve as a convenient "evaluation marker" for studying alterations in the biochemistry of the host, particularly with respect to environmental changes. In addition, the MTS-PMS colorimetric assay examination of antimicrobial activity appears to be superior to existing methods and may offer more general application in the search for new antibiotic molecules.
