Codon optimization for enhanced Escherichia coli expression of human S100A11 and S100A1 proteins
Codon optimization for enhanced Escherichia coli expression of human S100A11 and S100A1 proteins
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DOI：
10.1016/j.pep.2010.03.015
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发表时间：
2010-09-01
影响因子：
1.6
通讯作者：
Shaw, Gary S.
中科院分区：
文献类型：
--
作者：
Marlatt, Nicole M.;Spratt, Donald E.;Shaw, Gary S.
本文报道了重组全长人S100 A11和人S100 A1蛋白的克隆、表达和纯化。通过重叠不同长度的互补单链寡核苷酸合成基因。通过根据大肠杆菌偏好性仅选择最优选的密码子，对两种基因的编码序列进行密码子优化。为了将各种寡核苷酸组装成正确的全长基因，实施了独特的一步PCR程序。还对每种蛋白的表达和纯化程序进行了优化。单个苯基-琼脂糖凝胶柱足以纯化人S100 A11，而HiTrap Q阴离子交换随后苯基-琼脂糖凝胶柱用于纯化S100 A1。通过优化S100 A1和S100 A11基因、表达和纯化方案，每升培养基分别获得超过45和150 mg的纯化的人蛋白。通过SDS-PAGE和质谱法（MS）验证蛋白质身份，并通过NMR光谱法进一步表征。这些结果为大量表达和纯化人S100 A1和S100 A11蛋白以进行生物物理学鉴定建立了有效的方法。(C)2010年爱思唯尔公司All rights reserved.
The cloning, expression and purification for the recombinant full-length human proteins S100A11 and human S100A1 is described. The genes were synthesized by overlapping complementary single-stranded oligonucleotides of various lengths. The coding sequence for both genes were codon optimized by selecting only the most preferential codons according to the Escherichia coli bias. In order to assemble the various oligonucleotides into the correct full-length genes, a unique one-step PCR procedure was implemented. The expression and purification procedures were also optimized for each protein. A single phenyl-Sepharose column was sufficient for the purification of human S100A11 whereas HiTrap Q anion exchange followed by phenyl-Sepharose columns were required for the purification of S100A1. By optimizing the S100A1 and S100A11 gene, expression and purification protocols, more than 45 and 150 mg, respectively of the purified human proteins were obtained per litre of media. Protein identity was verified by both SDS-PAGE and mass spectrometry (MS) and further characterized by NMR spectroscopy. These results have established an efficient method for the expression and purification of large quantities of human S100A1 and S100A11 proteins for biophysical characterization. (C) 2010 Elsevier Inc. All rights reserved.