Qoronfleh M W, Ho T F, Brake P G, Banks T M, Pulvino T A, Wahl R C, Eshraghi J, Chowdhury S K, Ciccarelli R B, Jones B N
Department of Molecular and Cellular Biology, Sterling Winthrop Pharmaceuticals Research Division, Collegeville, PA 19426-0900, USA.
J Biotechnol. 1995 Apr 15;39(2):119-28. doi: 10.1016/0168-1656(94)00149-7.
Molecular analogs of amino acids can be incorporated into proteins. The amino acid analog selenomethionine (SeMet) has been shown to be efficiently incorporated into the proteins of growing Escherichia coli. SeMet-containing proteins are known to produce sufficiently strong anomalous scatter permitting the solution of the selenomethionyl crystal structure by multiwavelength anomalous diffraction (MAD) techniques. The recombinant protein chosen for these studies is mature, truncated neutrophil collagenase (rmNC-t). The rmNC-t protein is a monomer of 163 amino acid residues featuring one active site and two Met residues. We developed a T7 polymerase expression system allowing incorporation of SeMet into rmNC-t protein produced in E. coli. Substitution of Met with SeMet was accomplished by culturing E. coli DL41(DE3), a SeMet-tolerant strain with metA lesion, in a defined medium containing SeMet as the sole source of Met. The SeMet-labeled rmNC-t was isolated from inclusion bodies by solubilizing in urea, purified by anion column chromatography, and then refolded in the presence of Ca2+ and Zn2+. Analysis of SeMet-labeled rmNC-t demonstrated that Met replacement was 100%. Enzymatic characterization revealed no obvious differences in activity or inhibitor binding between rmNC-t and the SeMet-labeled product. We have produced pure, active SeMet-labeled rmNC-t in sufficient quantities for macromolecular crystallography studies.
氨基酸的分子类似物可以掺入蛋白质中。已证明氨基酸类似物硒代蛋氨酸（SeMet）能有效地掺入生长中的大肠杆菌的蛋白质中。已知含SeMet的蛋白质会产生足够强的异常散射，从而允许通过多波长异常衍射（MAD）技术解析硒代蛋氨酰晶体结构。这些研究中选择的重组蛋白是成熟的截短型中性粒细胞胶原酶（rmNC-t）。rmNC-t蛋白是一个由163个氨基酸残基组成的单体，具有一个活性位点和两个甲硫氨酸残基。我们开发了一种T7聚合酶表达系统，可将SeMet掺入大肠杆菌中产生的rmNC-t蛋白中。通过在含有SeMet作为唯一甲硫氨酸来源的限定培养基中培养大肠杆菌DL41(DE3)（一种具有metA损伤的耐SeMet菌株），实现了用SeMet替代甲硫氨酸。通过在尿素中溶解从包涵体中分离出SeMet标记的rmNC-t，通过阴离子柱色谱法纯化，然后在Ca2+和Zn2+存在下复性。对SeMet标记的rmNC-t的分析表明甲硫氨酸替代率为100%。酶学特性表明，rmNC-t与SeMet标记产物之间在活性或抑制剂结合方面没有明显差异。我们已经制备了足够数量的纯的、有活性的SeMet标记的rmNC-t，用于大分子晶体学研究。
