Expression and Purification of Recombinant α-Defensins and α-Defensin Precursors in Escherichia coli
Expression and Purification of Recombinant α-Defensins and α-Defensin Precursors in Escherichia coli
复制标题
DOI：
10.1007/978-1-60761-594-1_4
复制
发表时间：
2010-01-01
期刊：
影响因子：
--
通讯作者：
Ouellette, Andre J.
中科院分区：
文献类型：
--
作者：
Figueredo, Share;Mastroianni, Jennifer R.;Ouellette, Andre J.
α-防御素的重组表达可以在大肠杆菌中以有效水平获得。将扩增的α-防御素或前α-防御素编码cDNA序列定向克隆到pET-28 a表达载体的EcoRI和SalI位点之间，并在大肠杆菌中表达。coli BL 21 RIS细胞。通过加入50 mM异丙基β-D-1-硫代半乳糖吡喃糖苷3-6 h，诱导在营养丰富的液体培养基中指数生长的细胞表达重组蛋白。通过离心收集的细菌细胞在非还原条件下在6 M胍-HCl中裂解后，通过镍-NTA柱上的亲和色谱纯化与其6xHis-34氨基酸N-末端融合配偶体融合的表达的防御素。在表达的无Met肽的N末端引入的Met密码子提供了独特的CNBr切割位点，使得能够通过连续C18 RP-HPLC释放无辅助残基的α-防御素。通过MALDI-TOF质谱法确认C18 RP-HPLC纯化肽的分子量，并使用分析型RP-HPLC和酸-尿素聚丙烯酰胺凝胶电泳评估肽均一性。以这种方式制备的α-防御素在生物化学上等同于天然分子。
Recombinant expression of alpha-defensins can be obtained at efficient levels in Escherichia coli. Amplified alpha-defensin or pro-alpha-defensin coding cDNA sequences arc cloned directionally between EcoRI and SalI sites of the pET-28a expression vector and expressed in E. coli BL21 RIS cells. Cells growing exponentially in nutrient-rich liquid medium are induced to express the recombinant protein by addition of 50 mM isopropyl beta-D-1-thiogalactopyranoside for 3-6 h. After bacterial cells collected by centrifugation arc lysed in 6 M guanidine-HCl under non-reducing conditions, the expressed defensin fused to its 6xHis-34 amino acid N-terminal fusion partner is purified by affinity chromatography on nickel-NTA columns. A Met codon introduced at the N terminus of expressed Met-free peptides provides a unique CNBr cleavage site, enabling release of the alpha-defensin free of ancillary residues by sequential C18 RP-HPLC. Molecular masses of C18 RP-HPLC purified peptides are confirmed by MALDI-TOF mass spectrometry, and peptide homogeneity is assessed using analytical RP-H PLC and acid-urea polyacrylamide gel electrophoresis. alpha-Defensins prepared in this manner are biochemically equivalent to the natural molecules.