A novel method for purifying recombinant human host defense cathelicidin LL-37 by utilizing its inherent property of aggregation.
The importance of human LL-37 in host defense and innate immunity is well appreciated as reflected by an exponential increase of relevant literature in Pub-Med. Although several articles reported the expression and purification of this cathelicidin, some protocols suffered from low efficiency in enzyme cleavage of fusion proteins due to aggregation and poor separation of recombinant LL-37 from the carrier protein on reverse-phase HPLC. We present a new method for purifying LL-37 that avoids both problems. In this method, the fusion protein (a tetramer) purified by metal affinity chromatography was readily cleaved at a thrombin site 30-residue upstream of the LL-37 sequence. The released LL-37-containing fragment formed a large soluble aggregate (approximately 95 kDa) at pH approximately 7, allowing a rapid and clean separation from the carrier thioredoxin (approximately 14 kDa) by size-exclusion chromatography. Recombinant LL-37 was released from the isolated aggregate by chemical cleavage in 50% formic acid at 50 degrees C for 32 h. Due to a dramatic difference in retention time, recombinant LL-37 was well resolved from the S-Tag-containing peptide by RP-HPLC. Compared to previous procedures, the new method involves fewer steps and is highly reproducible. It increases peptide yield by 53%. NMR data support the aggregation of LL-37 into a tetramer with increase of pH as well as the feasibility of structural studies of an isotope-labeled antimicrobial peptide in the lipid micelle of dioctanoyl phosphatidylglycerol (D8PG) for the first time.
Li
Yifeng
Y
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA.
Li
Xia
X
Li
He
H
Lockridge
Oksana
O
Wang
Guangshun
G
D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics; D005561:Formates / Q000737:chemistry; D006801:Humans; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D013917:Thrombin / Q000737:chemistry; D054804:Cathelicidins
D023181:Antimicrobial Cationic Peptides; D005561:Formates; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; C030544:formic acid; D013917:Thrombin; D054804:Cathelicidins