Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158
Journal Title: Journal of Microbiology and Infectious Diseases - Year 2014, Vol 4, Issue 1
Abstract
Objective: Synthetic cationic antimicrobial peptide (SC-AMP) is an important and upcoming therapeutic molecule against conventional antibiotics. In this study, an attempt was made to purify the SC-AMP without the enzymatic cleavage of the affinity tag, by using an intein-based system. Methods: The intein sequence was amplified from pTYB11 vector using PCR methodologies and the N-terminal of intein was ligated with SC-AMP. The designed construct, intein-SC-AMP was cloned into MCS region of cold shock expression vector, pCOLDI and the recombinant peptide was purified on a chitin affinity column by cleaving intein with 50 mM DTT without applying enzymatic cleavage. Later the peptide was quantified and its antibacterial activity of the purified peptide was studied using well diffusion method. Results: Initially, intein-SC-AMP was expressed as a fusion protein in both IPTG inducible E. coli BL21(DE3) and salt inducible E. coli GJ1158. Single step purification using CBD (chitin binding domain) - intein tag in salt inducible E. coli GJ1158, yields the SC-AMP in the soluble form at a concentration of 208 mg/L. The antibacterial activity and minimal inhibitory concentration (MIC) of the purified SC-AMP was studied against both Gram positive and Gram negative mi croorganisms. Conclusion: For the first time, single step purification of soluble SC-AMP was carried out using chitin-binding domain affinity tag in salt inducible E. coli GJ1158 without an application of nzymatic cleavage. J Microbiol Infect Dis 2014;4(1): 13-19 Key words: Synthetic cationic antimicrobial peptide, intein sequence, pCOLDI, chitin affinity column, E. coli BL21(DE3) and E. coli GJ1158
Authors and Affiliations
K Seetha Ram, M Mary Kumari, S. Divya Sri, N Bhargava Ramudu, JB Peravali, KK Pulicherla
Keywords
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- EP ID EP121067
- DOI 10.5799/ahinjs.02.2014.01.0117
- Views 134
- Downloads 0
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