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

Related Articles

Orta Doğu Solunum Sendromu: MERS

Orta Doğu Solunum Sendromu: MERSOrta Doğu Solunum Sendromu coronovirüsü (MERS-CoV) yeni ortaya çıkan bir solunum virüsüdür. Bu virus ilk olarak 2012 yılında Suudi Arabistan’da tanımlanmıştır. Orta Doğu solunum sendromu k...

Effect of subinhibitory concentration of cefetoxime on biofilm formation

Objectives: In the present study, we have analyzed ESBL-producing S. typhi’s capability in forming a significant amount of biofilm on plastic and glass surface, and the influence of cefetoxime on biofilm development at s...

Development of Simplex-PCR Assays for Accurate Identification of Nine Staphylococcal Species at Genus and Species Levels

Development of Simplex-PCR Assays for Accurate Identification of Nine Staphylococcal Species at Genus and Species Levels Bibek Ranjan Shome1, Krithiga Natesan1, Susweta Das Mitra1,2, Nimita Venugopal1, Bhuvana Mani1, Fer...

Antimicrobial Efficacy of Silymarin and Silibinin Against Oral Microorganisms

Objective: To evaluate the antimicrobial effect of silymarin and silibinin, a plant derived flavonoid compounds against oral microorganisms that are responsible for dental caries. Methods: In the current investigation, w...

Surviving Mardi Gras in an Intensive Care Unit

Penicilliosis is a life threatening fungal infection caused by Penicillium marneffei (PM). It has a high mortality rate if not picked up in time and treated appropriately. Making an early diagnosis is a key in improving...

Download PDF file
  • EP ID EP121067
  • DOI 10.5799/ahinjs.02.2014.01.0117
  • Views 134
  • Downloads 0

How To Cite

K Seetha Ram, M Mary Kumari, S. Divya Sri, N Bhargava Ramudu, JB Peravali, KK Pulicherla (2014). Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158. Journal of Microbiology and Infectious Diseases, 4(1), 13-19. https://europub.co.uk/articles/-A-121067