Expression and Purification of the Antimicrobial Peptide Cecropin Ad by Fusion with Cationic Elastin-Like Polypeptides

Topics: Protein, Escherichia coli, DNA Pages: 10 (3749 words) Published: January 28, 2013
Protein Expression and Purification 85 (2012) 200–203

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Protein Expression and Purification
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Expression and purification of the antimicrobial peptide cecropin AD by fusion with cationic elastin-like polypeptides Ke Yang, Yujie Su, Junhua Li, Jun Sun, Yanjun Yang ⇑
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China

a r t i c l e

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a b s t r a c t
Cationic elastin-like polypeptides (CELP) are thermally responsive polypeptides that undergo an inverse temperature phase transition, and the recombinant CELP fusion proteins may be purified by inverse transition cycling (ITC). To obtain high-purity antimicrobial peptide cecropin AD (CAD), CELP was placed at the N-terminus of CAD and the expression vector pET28a-CELP-CAD was constructed. The expression vector was then transformed into Escherichia coli BL21 (DE3) to express the recombinant protein. After three rounds of ITC, enterokinase digestion and another hot spin, 1.2 mg recombinant CAD was purified from 100 ml culture medium. The antimicrobial 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 March 2012 and in revised form 11 April 2012 Available online 29 May 2012 Keywords: Antimicrobial peptide Cecropin AD Cationic elastin-like polypeptides Fusion expression

Introduction Cecropin AD (CAD)1, a cationic antimicrobial peptide (AMP), is composed of the first 11 residues of cecropin A and the last 26 ones of cecropin D [1]. CAD was found to have strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, which made itself attractive as a potential substitute of antibiotics [2]. Commercial applications require high-purity peptides to be readily available in an inexpensive manner. Chemical synthesis is a complicated and costly method, and CAD has been expressed and secreted through Pichia pastoris and Bacillus subtilis [2,3]; however, the isolation of CAD from fermentation liquid is still a time-consuming process [4]. Therefore, it is urgent to develop a simple and reliable method to obtain high-purity CAD. Fusion expression in Escherichia coli has been the most widely used method for the expression of AMPs [5]. The carrier proteins, fused to the AMPs, protect the host from the toxic peptides, and some of them may also be used as affinity tags [6]. Elastin-like polypeptides (ELP) consist of the repeating pentapeptides (Val-Pro-Gly-Xaa-Gly) where the residue Xaa as a guest residue represents any amino except proline [7]. ELP tags have revolutionized recombinant protein expression [8], due to the fact that they allow non-chromatographic purification by inverse transition cycling (ITC) [9]. Compared with those used ⇑ Corresponding author. Address: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, PR China. Fax: +86 510 85329080. E-mail address: (Y. Yang). 1 Abbreviations used: CELP, cationic elastin-like polypeptides; ITC, inverse transition cycling; CAD, cecropin AD; PBS, phosphate buffered saline; SUMO, small ubiquitinrelated modifier. 1046-5928/$ - see front matter Ó 2012 Published by Elsevier Inc.

for the fusion expression of AMPs, ELP make the isolation process more effective. The length of the ELP sequence was discovered to be influential on the yield [10], and the previously used ELP tag, composed of 110 pentapeptides in length [11], was not suitable as a carrier protein of small proteins like AMPs [12]. Lim [10] once reported that a 15.9 kDa cationic ELP (36 pentapeptides in length) with a guest residue composition of K:V:F of 1:7:1 was the optimal...

References: [1] B. Christensen, J. Fink, R.B. Merrifield, D. Mauzerall, Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes, Proc. Natl. Acad. Sci. USA 85 (1988) 5072–5076. [2] F.L. Jin, X.X. Xu, X.Q. Yu, S.X. Ren, Expression and characterization of antimicrobial peptide Cecropin AD in the methylotrophic yeast Pichia pastoris, Process Biochem. 44 (2009) 11–16. [3] X. Chen, F.M. Zhu, Y.H. Cao, S.Y. Qiao, Novel expression vector for secretion of cecropin AD in Bacillus subtilis with enhanced antimicrobial activity, Antimicrob. Agents Chem. 53 (2009) 3683–3689.
[4] S. Macauley-Patrick, M.L. Fazenda, B. McNeil, L.M. Harvey, Heterologous protein production using the Pichia pastoris expression system, Yeast 22 (2005) 249–270. [5] Y.F. Li, Recombinant production of antimicrobial peptides in Escherichia coli: a review, Protein Expression Purif. 80 (2011) 260–267. [6] Y.Q. Chen, S.Q. Zhang, B.C. Li, W. Qiu, B. Jiao, J. Zhang, Z.Y. Diao, Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners, Protein Expression Purif. 57 (2008) 303–311. [7] A. Chilkoti, T. Christensen, J.A. MacKay, Stimulus responsive elastin biopolymers: applications in medicine and biotechnology, Curr. Opin. Chem. Biol. 10 (2006) 652–657. [8] D.M. Floss, K. Schallau, S. Rose-John, U. Conrad, J. Scheller, Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application, Trends Biotechnol. 28 (2010) 37–45. [9] D.M. Floss, M. Sack, 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 a tobacco-derived human immunodeficiency virus-neutralizing antibody, Plant Biotechnol. J. 7 (2009) 899–913. [10] D.W. Lim, K. Trabbic-Carlson, J.A. MacKay, A. Chilkoti, Improved nonchromatographic purification of a recombinant protein by cationic elastinlike polypeptides, Biomacromolecules 8 (2007) 1417–1424. [11] M.R. Banki, L. Feng, D.W. Wood, Simple bioseparations using self-cleaving elastin-like polypeptide tags, Nat. Methods 2 (2005) 659–661. [12] Y. Shen, H.X. Ai, R. Song, Z.N. Liang, J.F. Li, S.Q. Zhang, Expression and purification of moricin CM4 and human beta-defensins 4 in Escherichia coli using a new technology, Microbiol. Res. 165 (2010) 713–718. [13] J. Arnau, C. Lauritzen, G.E. Petersen, J. Pedersen, Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins, Protein Expression Purif. 48 (2006) 1–13. [14] W. Cao, Y.X. Zhou, Y.S. Ma, Q.P. Luo, D.Z. Wei, Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli, Protein Expression Purif. 40 (2005) 404–410. [15] Z.N. Xu, L. Peng, Z.X. Zhong, X.M. Fang, P.L. Cen, High-level expression of a soluble functional antimicrobial peptide, human beta-defensin 2, in Escherichia coli, Biotechnol. Prog. 22 (2006) 382–386. [16] L. Wang, C.E. Lai, Q.F. Wu, J.L. Liu, M.J. Zhou, Z.H. Ren, D.D. Sun, S.W. Chen, A.L. Xu, Production and characterization of a novel antimicrobial peptide HKABF by Pichia pastoris, Process Biochem. 43 (2008) 1124–1131. [17] X.M. Lu, X.B. Jin, J.Y. Zhu, H.F. Mei, Y. Ma, F.J. Chu, Y. Wang, X.B. Li, Expression of the antimicrobial peptide cecropin fused with human lysozyme in Escherichia coli, Appl. Microbiol. Biotechnol. 87 (2010) 2169–2176. [18] F. Fan, Y.M. Wu, J.X. Liu, Expression and purification of two different antimicrobial peptides, PR-39 and Protegrin-1 in Escherichia coli, Protein Expression Purif. 73 (2010) 147–151. [19] T. Christensen, M. Amiram, S. Dagher, K. Trabbic-Carlson, M.F. Shamji, L.A. Setton, A. Chilkoti, Fusion order controls expression level and activity of elastin-like polypeptide fusion proteins, Protein Sci. 18 (2009) 1377–1387. [20] L. Li, J.X. Wang, X.F. Zhao, C.H. Kang, N. Liu, J.H. Xiang, F.H. Li, S. Sueda, H. Kondo, High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris, Protein Expression Purif. 39 (2005) 144–151. [21] B.A. Fong, W.Y. Wu, D.W. Wood, The potential role of self-cleaving purification tags in commercial-scale processes, Trends Biotechnol. 28 (2010) 272–279. [22] S.H. Shahravan, X.L. Qu, I.S. Chan, J.A. Shin, Enhancing the specificity of the enterokinase cleavage reaction to promote efficient cleavage of a fusion tag, Protein Expression Purif. 59 (2008) 314–319. [23] L. Huang, S.S.J. Leong, R.R. Jiang, Soluble fusion expression and characterization of bioactive human beta-defensin 26 and 27, Appl. Microbiol. Biotechnol. 84 (2009) 301–308. [24] M.P. Malakhov, M.R. Mattern, O.A. Malakhova, M. Drinker, S.D. Weeks, T.R. Butt, SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins, J. Struct. Funct. Genomics 5 (2004) 75–86.
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