Journal article
P-Link: A method for generating multicomponent cytochrome P450 fusions with variable linker length
RWTH Aachen University1
University of California at San Diego2
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark4
Bacterial Cell Factories, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark5
DECHEMA Research Institute6
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 P450(cin) 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-beta-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.
| Language: | English |
|---|---|
| Publisher: | Future Science Ltd |
| Year: | 2014 |
| Pages: | 13-20 |
| ISSN: | 19409818 and 07366205 |
| Types: | Journal article |
| DOI: | 10.2144/000114187 |
1,8-CINEOLE 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines 10064, Biochemistry studies - Proteins, peptides and amino acids 10802, Enzymes - General and comparative studies: coenzymes 2-beta-hydroxy-1,8-cineole BIOCHEMICAL BIOCHEMISTRY Biochemistry and Molecular Biophysics CinA protein C terminus, monooxygenase CinA-CinC fusion protein CinC flavodoxin shuttle protein ELECTRON-TRANSFER ESCHERICHIA-COLI EXPRESSION Enzymology HEME DOMAINS Methods and Techniques P-Link laboratory techniques, genetic techniques P450 ENZYMES P450(cin) PCR amplification polymerase chain reaction amplification laboratory techniques, genetic techniques PLICing PROTEIN REDOX PARTNER REDUCTASE DOMAIN SYSTEM amino acid linkers cinAgene cinCgene cloning laboratory techniques, genetic techniques cytochrome P450 9035-51-2 EC 1.14.14.1 directed evolution fusion protein fusion proteins linker monooxygenase multicomponent system protein engineering transformation laboratory techniques, genetic techniques
