Manifestation and Purification of the Anti-bacterial Peptide Cecropin Ad by Fusion with Cationic Elastin-Like Polypeptides

 Essay regarding Expression and Purification in the Antimicrobial Peptide Cecropin Advertising by Blend with Cationic Elastin-Like Polypeptides

Protein Phrase and Purification 85 (2012) 200–203

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Protein Manifestation and Purification

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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 [1]. 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 [2]. 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 [4]. 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 [5]. 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 [6]. 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 [7]. ELP tags include revolutionized recombinant protein phrase [8], due to the fact that they allow non-chromatographic purification simply by inverse move cycling (ITC) [9]. 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.

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 [10], as well as the previously used ELP tag, made up of 110 pentapeptides in length [11], had not been suitable as a carrier healthy proteins of little proteins like AMPs [12]. Lim [10] 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...

References: [1] B. Christensen, J. Fink, R. M. Merrifield, M. Mauzerall, Channel-forming properties of cecropins and related style compounds incorporated into planar lipid membranes, Proc. Natl. Acad. Sci. USA 85 (1988) 5072–5076. [2] F. L. Jin, X. Times. Xu, Times. Q. Yu, S. X. Ren, Expression and portrayal of antimicrobial peptide Cecropin AD in the methylotrophic yeast Pichia pastoris, Process Biochem. 44 (2009) 11–16. [3] X. Chen, F. Meters. Zhu, Y. H. Cao, S. Y. Qiao, Story expression vector for secretion of cecropin AD in Bacillus subtilis with increased antimicrobial activity, Antimicrob. Real estate agents Chem. 53 (2009) 3683–3689.

[4] S. Macauley-Patrick, Meters. L. Fazenda, B. McNeil, L. M. Harvey, Heterologous protein production using the Pichia pastoris appearance system, Candida 22 (2005) 249–270. [5] Y. Farrenheit. Li, Recombinant production of antimicrobial peptides in Escherichia coli: a review, Protein Phrase Purif. 85 (2011) 260–267. [6] Sumado a. Q. Chen, S. Q. Zhang, B. C. Li, W. Qiu, B. Jiao, J. Zhang, Z. Con. Diao, Appearance of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners, Protein Manifestation Purif. 57 (2008) 303–311. [7] A. Chilkoti, Capital t. Christensen, T. A. MacKay, Stimulus responsive elastin biopolymers: applications in medicine and biotechnology, Curr. Opin. Chem. Biol. 10 (2006) 652–657. [8] M. M. Get flossing, K. Schallau, S. Rose-John, U. Conrad, J. Scheller, Elastin-like polypeptides revolutionize recombinant protein appearance and their biomedical application, Developments Biotechnol. twenty-eight (2010) 37–45. [9] G. M. Floss their teeth, M. Bag, E. Arcalis, J. Stadlmann, H. Quendler, T. Rademacher, E. Stoger, J. Scheller, R. Fischer, U. Conrad, Influence of elastin-like peptide fusions on the quantity and quality of the tobacco-derived human being immunodeficiency virus-neutralizing antibody, Plant Biotechnol. J. 7 (2009) 899–913. [10] D. W. Lim, T. Trabbic-Carlson, M. A. MacKay, A. Chilkoti, Improved nonchromatographic purification of your recombinant proteins by cationic elastinlike polypeptides, Biomacromolecules eight (2007) 1417–1424. [11] M. R. Banki, L. Feng, D. W. Wood, Simple bioseparations using self-cleaving elastin-like polypeptide tags, Nat. Strategies 2 (2005) 659–661. [12] Y. Shen, H. Back button. Ai, L. Song, Z .. N. Liang, J. N. Li, H. Q. Zhang, Expression and purification of moricin CM4 and human being beta-defensins 5 in Escherichia coli utilizing a new technology, Microbiol. Res. 165 (2010) 713–718. [13] M. Arnau, C. Lauritzen, G. E. Petersen, J. Pedersen, Current methods for the use of affinity tags and tag removal for the purification of recombinant protein, Protein Phrase Purif. 48 (2006) 1–13. [14] Watts. Cao, Con. X. Zhou, Y. S. Ma, Queen. P. Luo, D. Z. Wei, Expression and purification of anti-bacterial peptide adenoregulin with C-amidated terminus in Escherichia coli, Protein Phrase Purif. forty five (2005) 404–410. [15] Unces. N. Xu, L. Peng, Z. By. Zhong, X. M. Fischzug, P. L. Cen, High-level expression of your soluble efficient antimicrobial peptide, human beta-defensin 2, in Escherichia coli, Biotechnol. Prog. 22 (2006) 382–386. [16] L. Wang, C. Elizabeth. Lai, Q. F. Wu, J. L. Liu, M. J. Zhou, Z. H. Ren, M. D. Sunlight, S. W. Chen, A. L. Xu, Production and characterization of your novel antimicrobial peptide HKABF by Pichia pastoris, Procedure Biochem. 43 (2008) 1124–1131. [17] Back button. M. Lu, X. N. Jin, J. Y. Zhu, H. Farrenheit. Mei, Y. Ma, Farreneheit. J. Chu, Y. Wang, X. N. Li, Appearance of the anti-bacterial peptide cecropin fused with human lysozyme in Escherichia coli, Appl. Microbiol. Biotechnol. 87 (2010) 2169–2176. [18] F. Supporter, Y. M. Wu, M. X. Liu, Expression and purification of two distinct antimicrobial peptides, PR-39 and Protegrin-1 in Escherichia coli, Protein Expression Purif. 73 (2010) 147–151. [19] T. Christensen, Meters. Amiram, S i9000. Dagher, T. Trabbic-Carlson, Meters. F. Shamji, L. A. Setton, A. Chilkoti, Fusion order controls expression level and activity of elastin-like polypeptide fusion aminoacids, Protein Sci. 18 (2009) 1377–1387. [20] L. Li, J. By. Wang, Back button. F. Zhao, C. They would. Kang, N. Liu, J. H. Xiang, F. H. Li, S i9000. Sueda, They would. Kondo, High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris, Protein Manifestation Purif. 39 (2005) 144–151. [21] N. A. Fong, W. Con. Wu, G. W. Real wood, The potential role of self-cleaving purification tags in commercial-scale processes, Styles Biotechnol. twenty eight (2010) 272–279. [22] S. H. Shahravan, X. D. Qu, We. S. Chan, J. A. Shin, Improving the specificity of the enterokinase cleavage reaction to promote efficient cleavage of the fusion indicate, Protein Expression Purif. fifty nine (2008) 314–319. [23] L. Huang, S. S. J. Leong, R. R. Jiang, Soluble blend expression and characterization of bioactive individual beta-defensin 21 and twenty seven, Appl. Microbiol. Biotechnol. 84 (2009) 301–308. [24] M. P. Malakhov, M. Ur. Mattern, U. A. Malakhova, M. Consumer, S. D. Weeks, Capital t. R. Butt, SUMO liquidation and SUMO-specific protease pertaining to efficient phrase and purification of healthy proteins, J. Struct. Funct. Genomics 5 (2004) 75–86.