Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia co
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APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities Rasha Abou Aleinein & Razan Hamoud & Holger Schäfer & Michael Wink
Received: 6 June 2012 / Revised: 22 August 2012 / Accepted: 16 September 2012 # Springer-Verlag Berlin Heidelberg 2012
Abstract The coding sequence, which corresponds to the mature antimicrobial peptide ranalexin from the frog Rana catesbeiana, was chemically synthesized with preferred codons for expression in Escherichia coli. It was cloned into the vector pET32c (+) to express a thioredoxin-ranalexin fusion protein which was produced in soluble form in E. coli BL21 (DE3) induced under optimized conditions. After two purification steps through affinity chromatography, about 1 mg of the recombinant ranalexin was obtained from 1 L of culture. Mass spectrometrical analysis of the purified recombinant ranalexin demonstrated its identity with ranalexin. The purified recombinant ranalexin is biologically active. It showed antibacterial activities similar to those of the native peptide against Staphylococcus aureus, Streptococcus pyogenes, E. coli, and multidrug-resistant strains of S. aureus with minimum inhibitory concentration values between 8 and 128 μg/ml. The recombinant ranalexin is also cytotoxic in HeLa and COS7 human cancer cells (IC50 013–15 μg/ml). Keywords Ranalexin . Fusion protein . Antimicrobial peptide . Escherichia coli . Recombinant protein
Introduction Since the 1970s, methicillin-resistant Staphylococcus aureus (MRSA) has become a major cause of worldwide hospital infections which have significantly increased in recent years R. A. Aleinein : R. Hamoud : H. Schäfer : M. Wink (*) Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany e-mail: [email protected]
(Berger-Bachi and Rohrer 2002; David and Daum 2010). Therefore, scientists have paid much attention to the development of novel antibiotics, among them antimicrobial peptides (AMPs) as a potential source of active natural products (Maloy and Kari 1995; Sader et al. 2007; Schwab et al. 1999). AMPs are small peptides, generally composed of 12–60 amino acids. They are considered as an evolutionarily ancient component of the innate immune response against microbes in many organisms (Hancock 1997; Zasloff 2002). AMPs are widely distributed among amphibians, mollusks, higher vertebrates, and even plants (Hancock 2001). AMPs exhibit a broad spectrum of activities against bacteria, fungi, parasites, viruses, and even some tumor cells (Conlon et al. 2004) at a concentration which is relatively nontoxic to eukaryotic cells and which makes AMPs attractive candidates as novel therapeutic agents. The likely mode of antimicrobial activity derives from electrostatic interactions between AMPs and the negatively charged phospholipids in bilayer membranes which cause “barrel stave,” “toroidal pores,” or destroy the memb
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