Active, soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation

Abstract

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 approximately 0.5 to 1 mg/L of E. coli culture.

Melittin–glutathione S-transferase fusion protein exhibits anti-inflammatory properties and minimal toxicity

“…It has been shown that melittin, in its native form, at concentrations under 3 μM has anti-inflammatory properties, but validation of the toxicity at these concentrations toward the cell-lines was not apparent (Mosser and Edwards, 2008; Park et al, 2004; Park et al, 2007). We have observed that melittin, in its native form, has toxicity at concentrations in the nanomolar range in macrophages having previously shown similar toxicity in bacteria and cancer lines (Buhrman et al, 2013a; Buhrman et al, 2013b; Buhrman et al, 2013c; Buhrman et al, 2012). This is consistent with several studies in other cell types (Nah et al, 2007; Nishikawa and Kitani, 2011; Soman et al, 2009).…”

“…After cleavage of the GST from the melittin via a thrombin-cleavable site (Buhrman et al, 2013b; Buhrman et al, 2013c), we show decreases in cellular viability, interpreted as cell death, similar to the native peptide at these concentrations ( Figure 1C ). Similarly, we show that native peptide with the addition of free GST maintains ability to kill ( Figure 1D ).…”

“…Therefore, we sought to explore methods to improve the therapeutic response of the melittin peptide while maintaining its therapeutic effects. While examining the use of GST-GSH interactions for controlling the release of melittin (Buhrman et al, 2013b; Buhrman et al, 2013c; Buhrman et al, 2012), we observed the diminished toxicity of GST-melittin fusion protein. We validated this finding in macrophages at fusion protein concentrations up to 30 μM.…”

“…GST-melittin was purified by a method previously described (Buhrman et al, 2013c). The expression of both GST and GST-melittin was induced in the Rosetta strain (Novagen/EMD Millipore, Billerica, MA).…”

“…GST was purified from the soluble fraction by Ni-NTA agarose (Qiagen, Venlo, NLD) according to the manufacturer's instructions. GST-melittin was extracted from the insoluble fraction by the acidic extraction method previously reported (Buhrman et al, 2013c; Buhrman et al, 2012). Briefly, the insoluble lysate was washed with 70 mM TCEP (Thermo-Fisher Scientific) in PBS (pH = 2.3), and GST-melittin was then extracted in PBS with 70 mM TCEP and 1% tween 20 (Thermo-Fisher Scientific).…”