Department of Chemistry, Technische Universität Berlin, Müller-Breslau-Strasse 10, 10623 Berlin, Germany; Institute of Chemistry and Biochemistry - Organic Chemistry, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany.
Department of Chemistry, Technische Universität Berlin, Müller-Breslau-Strasse 10, 10623 Berlin, Germany.
Enzyme Microb Technol. 2017 Nov;106:55-59. doi: 10.1016/j.enzmictec.2017.06.014. Epub 2017 Jun 29.
Global substitution of canonical amino acids (cAAs) with noncanonical (ncAAs) counterparts in proteins whose function is dependent on post-translational events such as cofactor binding is still a methodically challenging and difficult task as ncAA insertion generally interferes with the cofactor biosynthesis machinery. Here, we report a technology for the expression of fully substituted and functionally active cofactor-containing hemeproteins. The maturation process which yields an intact cofactor is timely separated from cAA→ncAA substitutions. This is achieved by an optimised expression and fermentation procedure which includes pre-induction of the heme cofactor biosynthesis followed by an incorporation experiment at multiple positions in the protein sequence. This simple strategy can be potentially applied for engineering of other cofactor-containing enzymes.
在依赖于翻译后事件（如辅助因子结合）的蛋白质中，将规范氨基酸（cAAs）全局替换为非规范（ncAAs）对应物仍然是一项具有挑战性和困难的任务，因为 ncAA 插入通常会干扰辅助因子生物合成机制。在这里，我们报告了一种表达完全取代和功能活性的含辅助因子血红素蛋白的技术。产生完整辅助因子的成熟过程及时与 cAA→ncAA 取代分离。这是通过优化的表达和发酵程序实现的，该程序包括预先诱导血红素辅助因子生物合成，然后在蛋白质序列的多个位置进行掺入实验。这种简单的策略可潜在地应用于其他含辅助因子酶的工程改造。
