Manifestation and Purification of the Anti-bacterial Peptide Cecropin Ad by Fusion with Cationic Elastin-Like Polypeptides
Protein Phrase and Puriﬁcation 85 (2012) 200–203
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Protein Manifestation and Puriﬁcation
journal home page: www.elsevier.com/locate/yprep
Expression and puriﬁcation of the antimicrobial peptide cecropin AD by simply fusion with cationic elastin-like polypeptides Ke Yang, Yujie Su, Junhua Li, Jun Sun, Yanjun Yang ⇑
State Important Laboratory of Food Research and Technology, Jiangnan College or university, Wuxi, Jiangsu 214122, PR China University of Foodstuff Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR Chinese suppliers
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Cationic elastin-like polypeptides (CELP) will be thermally responsive polypeptides that undergo an inverse temperatures phase change, and the recombinant CELP fusion proteins may be puriﬁed simply by inverse changeover cycling (ITC). To obtain high-purity antimicrobial peptide cecropin ADVERTISING (CAD), CELP was put at the N-terminus of CAD and the manifestation vector pET28a-CELP-CAD was constructed. The expression vector was then simply transformed into Escherichia coli BL21 (DE3) to convey the recombinant protein. Following three models of ITC, enterokinase digestion and one more hot spin, 1 . 2 mg recombinant CAD was puriﬁed by 100 ml culture medium. The anti-bacterial test indicated that the high-purity CAD had strong antimicrobial activity against E. coli and Staphylococcus aureus. Ó 2012 Published by Elsevier Inc.
Article history: Received 31 Mar 2012 and in revised contact form 11 April 2012 Available 29 May possibly 2012 Keywords: Antimicrobial peptide Cecropin AD Cationic elastin-like polypeptides Blend expression
Intro Cecropin ADVERTISEMENT (CAD)1, a cationic antimicrobial peptide (AMP), is composed of the ﬁrst 10 residues of cecropin A and the previous 26 ones of cecropin D . CAD was found to have good antimicrobial activity against equally Gram-positive and Gram-negative bacteria, which produced itself attractive as a potential substitute of antibiotics . Business applications require high-purity peptides to be easily available in an economical manner. Chemical substance synthesis can be described as complicated and costly technique, and CAD has been portrayed and released through Pichia pastoris and Bacillus subtilis [2, 3]; yet , the isolation of CAD from fermentation liquid is still a time-consuming procedure . Therefore , it really is urgent to produce a simple and reliable approach to obtain high-purity CAD. Fusion expression in Escherichia coli has been the most favored method for the expression of AMPs . The company proteins, fused to the Amplifiers, protect the host in the toxic peptides, and some of them may also be used as afﬁnity tags . Elastin-like polypeptides (ELP) consist of the echoing pentapeptides (Val-Pro-Gly-Xaa-Gly) where the deposits Xaa like a guest residue represents any kind of amino except proline . ELP tags include revolutionized recombinant protein phrase , due to the fact that they allow non-chromatographic puriﬁcation simply by inverse move cycling (ITC) . Compared with those used ⇑ Corresponding writer. Address: Express Key Lab of Meals Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China and tiawan. Fax: +86 510 85329080. E-mail addresses: [email protected] com (Y. Yang). 1 Short-hand used: CELP, cationic elastin-like polypeptides; ITC, inverse move cycling; CAD, cecropin AD; PBS, phosphate buffered saline; SUMO, small ubiquitinrelated modiﬁer. 1046-5928/$ - see the front matter Ó 2012 Printed by Elsevier Inc. http://dx.doi.org/10.1016/j.pep.2012.04.007
for the fusion phrase of Amplifiers, ELP associated with isolation process more effective. The length of the ELP sequence was discovered to be inﬂuential for the yield , as well as the previously used ELP tag, made up of 110 pentapeptides in length , had not been suitable as a carrier healthy proteins of little proteins like AMPs . Lim  once reported which a 15. on the lookout for kDa cationic ELP (36 pentapeptides in length) using a guest deposits composition of K: Sixth is v: F of just one: 7: one particular was the optimal...
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