Swiderska K W, Szlachcic A, Czyrek A, Zakrzewska M, Otlewski J
Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland.
Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wroclaw, Poland.
Bioorg Med Chem. 2017 Jul 15;25(14):3685-3693. doi: 10.1016/j.bmc.2017.05.003. Epub 2017 May 5.
Recent advances in site-specific protein modification include the increasingly popular incorporation of unnatural amino acid(s) using amber codon, a method developed by Schultz and coworkers. In this study, we employ this technique to introduce propargyllysine (PrK) in human fibroblast growth factor 2 (FGF2). Owing to an alkyne moiety in its side chain, PrK is compatible with Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC). We successfully tested CuAAC-mediated conjugation of FGF2 with two compounds - a fluorophore carboxyrhodamine 110 or a cytotoxic drug monomethyl auristatin E (MMAE). In the case of the MMAE conjugate we improved the initial poor conjugation yield to achieve nearly 100% efficiency after extensive optimization. The detergent-based optimization approach may help overcome problems with the CuAAC reaction yield for protein modification with hydrophobic compounds, such as MMAE.
位点特异性蛋白质修饰的最新进展包括使用琥珀密码子引入非天然氨基酸的方法越来越流行，该方法由舒尔茨及其同事开发。在本研究中，我们采用该技术在人成纤维细胞生长因子2（FGF2）中引入炔丙基赖氨酸（PrK）。由于其侧链中的炔基部分，PrK与铜（I）催化的叠氮化物-炔烃1,3-偶极环加成反应（CuAAC）兼容。我们成功测试了CuAAC介导的FGF2与两种化合物的共轭反应——一种荧光团羧基罗丹明110或一种细胞毒性药物单甲基奥瑞他汀E（MMAE）。在MMAE共轭物的情况下，我们改善了最初较差的共轭产率，经过广泛优化后实现了近100%的效率。基于去污剂的优化方法可能有助于克服使用疏水性化合物（如MMAE）进行蛋白质修饰时CuAAC反应产率的问题。
