A method for generating multicomponent cytochrome P450 fusions with variable linker length

K. D. Belsare, A. J. Ruff, R. Martinez, A. V. Shivange, H. Mundhada, D. Holtmann, J. Schrader, U. Schwaneberg

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 gener-ating fusion proteins with variable linker lengths,
protein fusion with variable linker insertion (P-LinK),which was validated by fusing P450
cinmonooxygenase (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-b-hydroxy-1,8-cin-
eole 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.
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