Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. any further purification using this technique. Detergents were removed with a final purification on nickel agarose to achieve a final protein yield of 5 to 10 mg/L of culture. This corresponds to a real melittin yield of Dexamethasone tyrosianse inhibitor 0.5 to 1 1.0 mg/liter of culture after removal of the GST tag. Further, we confirm that recombinant melittin is similar to synthetic melittin in terms of cell lysis using in two very different organisms: U-87 MG human malignancy cells and bacteria. We show, in these studies, that recombinant melittin is effective at inhibiting growth of both U-87 MG cells and pathogenic bacteria. We propose that this relatively high yield method of purifying functional melittin will make the potential drug more accessible for study and formulation. Methods Cloning of melittin gene into expression vector Melittin was cloned using standard cloning procedures. All restriction enzymes were bought from New Britain Biolabs, MA. The melittin peptide was designed as reported (GIGAVLKVLTTGLPALISWIKRKRQ).1 The rDNA was codon optimized with the JCAT codon optimization tool:9 AGC GGA TCC GGT ATC GGT GCT GTT CTG AAA GTT CTG ACC ACC GGT CTG CDC42 CCG GCT CTG ATC TCT TGG ATC AAA CGT AAA CGT CAG TAG GAA TTC CG. Limitation sites BamHI (dual underlined) and EcoRI (underlined) had been engineered towards the 5′ and 3′ ends respectively, and an amber end codon (TAG, italicized and underlined) was built at the 3′ end. This double-stranded fragment was synthesized by Integrated DNA technologies (Skokie, IL) and was cloned into the pJB-HTS variant of the pGEX6p-1 expression vector (GE Healthcare Biosciences, Pittsburgh, PA)10 generating pJB-HTS-melittin. Positive clones were screened Dexamethasone tyrosianse inhibitor by direct sequencing (ACGT, Wheeling, IL). The layout of the expected protein is usually N-GST-6xHis-thrombin cleavage site-melittin-C thus allowing dual purification with glutathione or nickel columns (Physique 1A). Open in a separate window Physique 1 Purification of GST-6xHis-melittin from soluble protein fraction(A) Predicted structure of GST-6xHis-melittin fusion protein based upon template matching.11 Approximate molecular weights are denoted below fragments as they would be generated by thrombin cleavage following the sequence LVPR. (B) GST-6xHis-melittin (orange and magenta arrow) was induced with 1 mM IPTG for 16 hours (lane 1) or 3 hours (lane 2) and compared with 200 ng of bovine serum albumin (BSA; lane 3), and a protein ladder for size (L). (C) GST-6xHis-melittin was also induced for 16 hours at 37C (lanes 1 and 2) or 25C (lanes 3 and 4) with 0.1 mM (lane 1 and Dexamethasone tyrosianse inhibitor 3) or 0.01 mM IPTG (lane 2 and 4). (D) The induced GST-6xHis-melittin has two purified products of the expected sizes for the fusion protein and GST-6xHis (orange arrow). Upon addition of 2U thrombin (+), the GST-6xHis-melittin was cleaved and only the GST-6xHis band was observed. In each panel, the orange and magenta arrow indicates GST-6xHis-melittin and the orange arrow indicates GST-6xHis. (For interpretation of the recommendations to color in this physique legend, the reader is referred to the web version of this article.) Expression and purification of Melittin GST-6xHis-melittin made up of plasmid (pJB-HTS-melittin) was transformed into competent Rosetta 2 cells (Novagen, Darmstadt, Germany) in order to negate the truncating effects of underrepresented codons within restriction sites around the pJB-HTS vector, upstream of the melittin insertion (CTC, AGA, GGA) and to eliminate reduced expression effects of the outer membrane protease T and Lon protease.12, 13 Cells from saturated, overnight starter cultures were incubated at 37C at 180 revolutions per minute (RPM) until the desired cell density (A600 ~ 0.4) before addition of IPTG, 1 mM , 0.1 mM, or 0.01 mM, and removal to the appropriate induction temperature, 37C, 25C, or 4C. After induction for varying occasions, 3 or 16 hours, cells were collected by centrifugation at 3600g and 4C for 20 moments and were resuspended in lysis buffer (50 mM NaHPO4, 300 mM NaCl, 10 mM imidazole, buffered at pH=8.0). Lysozyme (1 mg, Sigma-Aldrich, St. Louis,.