ISSN: 1314-6246 Perevali et al. J. BioSci. Biotech. 2013, 2(3): 181-188. RESEARCH ARTICLE http://www.jbb.uni-plovdiv.bg 181 Jawahar Babu Peravali 1† Seetha Ram Kotra 2† Gogata Ravi Teja 3 Prudvi Nelavalli 2 Kona Prasad 3 Anmol Kumar 2 Kunkalaguntla V. Rajesh 1 Kota Sobha 3 Ruther Nelson 4 Krishna Kanth Pulicherla 5 Molecular cloning, expression and in vitro analysis of soluble cationic synthetic antimicrobial peptide from salt-inducible Escherichia coli GJ1158 Authors’ address: 1 Department of Biotechnology, Bapatla Engineering College, Bapatla - 522101, Guntur, Andhra Pradesh, India. 2 Department of Biotechnology, Acharya Nagarjuna University, Guntur - 522510, Andhra Pradesh, India. 3 Department of Biotechnology, R.V.R. & J.C. College of Engineering, Chowdavaram, Guntur - 522019, Andhra Pradesh, India. 4 Department of Botany, Govt. Arts Colelge, Ariyalur, Tamilnadu, India. 5 CBST, VIT, Vellore, Tamilnadu, India. † JB Perevali and SR Kotra contributed equally to this work. Correspondence: JB Peravali Department of Biotechnology, Bapatla Engineering College, Bapatla - 522101, Guntur, Andhra Pradesh, India. Tel.: +91-9494152489 e-mail: jbperavali@gmail.com Article info: Received: 19 July 2013 In revised form: 31 August 2013 Accepted: 2 September 2013 ABSTRACT Antimicrobial peptides are the upcoming therapeutic molecules as alternative drugs to the existing antibiotics owing to their potent action against pathogenic microorganisms. In this study, to obtain an antimicrobial peptide with a broad range of activity, the synthetic cationic antimicrobial peptide was designed by using in silico tools viz., antimicrobial peptide database, protparam, hierarchical neural network. Later, the peptide was translated back into a core nucleotide sequence and the gene for the peptide was constructed by overlapping PCR. The amplified gene was cloned into pRSET–A vector and transformed into salt inducible expression host E. coli GJ1158. The expression results show high yields of soluble recombinant fusion peptide (0.52 g/L) from salt-inducible E. coli. The recombinant peptide was purified by the IMAC purification system and cleaved by enterokinase. The digested product was further purified and 0.12 g/L of biologically active recombinant cationic antimicrobial peptide was obtained. In vitro analysis of the purified peptide demonstrated high antimicrobial activity against both Gram positive and Gram negative bacteria devoid of hemolytic activity. Therefore, this synthetic cationic antimicrobial peptide could serves as an promising agent over chemical antibiotics. In this study, a synthetic cationic antimicrobial peptide was designed, cloned and expressed from salt-inducible E. coli GJ1158 using cost effective media in the large scale production of antimicrobial peptide and its biological activity was analysed against different Gram positive and negative organisms. Key words: synthetic antimicrobial peptide, in silco techniques, pRSET-A, IMAC purification, entirokinase, antimicrobial activity Introduction The discovery, development and clinical exploitation of antibiotics are one of the most significant medical advances in twentieth century (Hancock, 1997). However, the usage of antibiotics towards potential pathogens have been over used or used improperly, owing to resistance or multidrug resistance, which is emerged rapidly in many microbial species (Robert & Moellering, 2003). The pharmaceutical industry has continuously met this need by modifying the existing antibiotics. Despite the success to date in antimicrobial development, the inexorable, ongoing