Active, soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation.
Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U-87 MG glioma cells and inhibits growth of the gram-positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of ∼ 0.5-1 mg/L of E. coli culture.
Dept. of Biopharmaceutical Sciences, University of Illinois, Chicago, IL, 60612-7231.
Jason S
JS
Buhrman
Jamie E
JE
Rayahin
Laura C
LC
Cook
Michael J
MJ
Federle
Richard A
RA
Gemeinhart
D000900:Anti-Bacterial Agents / Q000302:isolation & purification; D000970:Antineoplastic Agents / Q000302:isolation & purification; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008555:Melitten / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D013297:Streptococcus pyogenes / Q000187:drug effects
D000900:Anti-Bacterial Agents; D000970:Antineoplastic Agents; D011994:Recombinant Proteins; D008555:Melitten
fusion protein; melittin; protein extraction; recombinant protein; surfactant