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...
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