Abstract:Melittin is one kind of small molecule active peptide with anti-inflammatory and antitumor effects. The soluble expression and purification of melittin to obtain active peptide without fusion label has always been a problem. The authors conducted a recombinant expression strain Escherichia coli/pET21a-ELP-Intein-MET based on the phase transition property of elastin-like polypeptide (ELP) and the self-cracking property of intein. Soluble expression of fusion protein was achieved in E. coli BL21 (DE3) cells. The supernatant of the recombinant cells was purified by a temperature-induced reversible phase transformation cycle, and optimization of pH, temperature and time of the fusion protein self-lysis. The melittin without fusion label with a purity of over 95% was obtained after lysis at pH 7.0 and 30 ℃ for 32 h, and the following gel chromatography. The inhibition experiments of purified melittin against Staphylococcus aureus, Bacillus subtilis 168 and Escherichia coli JM109 were performed. Melittin showed a stronger inhibitory effect for gram-positive bacteria than gram-negative bacteria. The minimum inhibitory concentrations of melittin for 3 strains of bacteria were 12.5 μg/mL, 50 μg/mL and 100 μg/mL, respectively. The authors realized the high expression of melittin by the introduction of elastin and endopeptide, and non-fusion labelled melittin was purified by a simple isolation. The bacteriostatic functions of purified active melittin were very similar to that of commercial melittin, which is expected to provide new insights for the industrial preparation of melittin.
