Characterization of factors favoring the expression and purification of recombinant LL-37 from Escherichia coli

Abstract

The only human antimicrobial peptide, LL-37, was overexpressed in soluble form using Escherichia coli-based expression system. Recombinant LL-37 production in E. coli was optimized for use in large quantities for studying antimicrobial activity against Helicobacter pylori strains. We previously reported a method to express and purify LL-37 using Glutathione S-transferase (GST) fusion system. Herein presents method suitable for producing recombinant LL-37 from the recombinant GST-LL-37 fusion protein by recovering from both soluble protein fractions and inclusion bodies. Compared to the yield reported previously, the yield of recombinant GST-LL-37 protein was much improved to 10 mg/L of culture by affinity chromatography using GSTrap FF (1 mL) affinity chromatography column. These results suggest that the production method used in present study is useful in obtaining large quantity of recombinant LL-37 peptide. The optimized recovery protocol from inclusion bodies was highly contributable in raising yield of GST-LL-37 fusion protein. When treated with Factor Xa, GST-LL-37 fusion protein recovered from both soluble protein fractions and inclusion bodies released an approximate 4.5-kDa protein, which was the expected size of LL-37; GST-LL-37 fusion protein recovered from both soluble protein fractions and inclusion bodies was also confirmed by enzymatic digestion with thrombin, which produced LL-37 peptide containing six extra amino acid residues, Gly-Ile-Ile-Glu-Gly-Arg, on Nterminus of LL-37. Purified recombinant LL-37 showed nearly identical antimicrobial activities against H. pylori strains as that of synthetic LL-37, suggesting its application to functional study with therapeutic potential on gastric pathogen.