Belsare Ketaki D, Ruff Anna Joëlle, Martinez Ronny, Shivange Amol V, Mundhada Hemanshu, Holtmann Dirk, Schrader Jens, Schwaneberg Ulrich
Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.
Department of Chemical Engineering, University of California, Santa Barbara, California.
Biotechniques. 2014 Jul 1;57(1):13-20. doi: 10.2144/000114187. eCollection 2014 Jul.
Fusion protein construction is a widely employed biochemical technique, especially when it comes to multi-component enzymes such as cytochrome P450s. Here we describe a novel method for generating fusion proteins with variable linker lengths, protein fusion with variable linker insertion (P-LinK), which was validated by fusing P450cin monooxygenase (CinA) to the flavodoxin shuttle protein (CinC). CinC was fused to the C terminus of CinA through a series of 16 amino acid linkers of different lengths in a single experiment employing 3 PCR amplifications. Screening for 2-β-hydroxy-1,8-cineole production by CinA-CinC fusion proteins revealed that enzymatically active variants possessed linker lengths of more than 5 amino acids, reaching optimum enzyme activity at a linker length of 10 amino acids. Our P-LinK method not only minimizes experimental effort and significantly reduces time demands but also requires only a single cloning and transformation step in order to generate multiple linker variants (1 to 16 amino acids long), making the approach technically simple and robust.
融合蛋白构建是一种广泛应用的生化技术，尤其是在涉及多组分酶如细胞色素P450时。在此，我们描述了一种生成具有可变连接子长度的融合蛋白的新方法，即可变连接子插入的蛋白融合（P-LinK），该方法通过将P450cin单加氧酶（CinA）与黄素氧还蛋白穿梭蛋白（CinC）融合进行了验证。在一次使用3次PCR扩增的实验中，通过一系列不同长度的16个氨基酸连接子，将CinC融合到CinA的C末端。对CinA-CinC融合蛋白产生2-β-羟基-1,8-桉叶素的筛选表明，具有酶活性的变体的连接子长度超过5个氨基酸，在连接子长度为10个氨基酸时达到最佳酶活性。我们的P-LinK方法不仅将实验工作量减到最小并显著减少了时间需求，而且只需要一个克隆和转化步骤就能生成多个连接子变体（1至16个氨基酸长），使得该方法在技术上简单且可靠。
