Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158

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 microorganisms. 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 enzymatic cleavage.

Recent Advances in Antibacterial and Antiendotoxic Peptides or Proteins from Marine Resources

“…As seen in the examples of NZ17074 and N6, SUMO is successfully applied to increase the solubility and yield of peptides [ 37 , 67 ]. Additionally, improvement in solvent extraction techniques (such as supercritical fluid extraction, pressurized solvent extraction and microware/ultrasound/pulsed electric field/enzyme-assisted extraction) and the cost–effective purification methods including using monolithic columns and the intein system in AMP production can also be introduced to reduce the production cost for antibacterial/antiendotoxic peptides or proteins ( Table 4 ) [ 17 , 105 , 106 , 107 ].…”

Site Directed Mutagenesis , Molecular Cloning and Expression of interleukin - 17E to Generate Structural Variant with Enhanced Specific Activity Using Industrial Friendly Salt Inducible Escherichia coli GJ1158

“…After confirmation, IL-17E structural variant was incubated in hydroxylamine cleavage buffer (2 M hydroxylamine hydrochloride, 0.2 M Tris HCl, pH 9.0) for 3 hrs at 42 -45 0 C [15]. The reaction was terminated by cooling the samples to room temperature.…”