Recombinant expression, isotope labeling, refolding, and purification of an antimicrobial peptide, piscidin.
An antimicrobial peptide, piscidin, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The peptide was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The expression and purification process of piscidin encountered several problems such as the lysis of the bacterial cell upon induction of the peptide production, the unwanted cleavage of the fusion protein inside the bacterial cell, and high tendency to aggregate in the aqueous environment. Such problems were alleviated by employing ubiquitin as a fusion partner for piscidin, growing the cells at a lower temperature, and changing the order of the purification steps. The yields of the fusion protein and the peptide were around 15 and 1.5 mg per liter of LB or minimal medium, respectively. The recombinant expression and purification of piscidin will enable its structural and dynamic studies using multidimensional NMR spectroscopy.
Moon
Won Jae
WJ
Department of Chemistry and Recombinant Protein Expression Center, Sejong University, 98 Gunja-Dong, Gwangjin-Gu, Seoul 143-747, Republic of Korea.
Hwang
Dong Kyu
DK
Park
Eu Jin
EJ
Kim
Yang Mee
YM
Chae
Young Kee
YK
D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000378:metabolism; D029941:Fish Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015870:Gene Expression; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D017510:Protein Folding; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D029941:Fish Proteins; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; C449802:moronecidin protein, Morone saxatilis