Phosphorothioate-based DNA recombination: an enzyme-free method for the combinatorial assembly of multiple DNA fragments

BioTechniques

Jan Marienhagen¹  Ulrich Schwaneberg¹  Alexander Dennig¹ 

[1] 1Department of Biotechnology, RWTH Aachen University, Aachen, Germany;

关键词: recombination;    directed evolution;    protein engineering;    phosphorothioate;    PTRec;   

DOI  :  10.2144/000113865

来源: DOAJ

【 摘 要 】

Rational guided generation of protein chimeras has developed into an attractive approach in protein engineering for tailoring catalytic and biophysical properties of enzymes. Combinatorial recombination of structural elements or whole protein domains is still technically challenging due to sequence dependent biases diminishing the overall quality of resulting chimeric libraries. Since methods for generating such libraries are often limited by a low frequency of crossover points and suffer from challenges in handling, we developed the phosphorothioate-based DNA recombination method (PTRec). PTRec is an enzyme-free method and only requires a short stretch of four amino acids that is identical among the proteins to be recombined in order to define a single crossover point. In a PTRec-generated chimeric library that shuffled five domains of phytase using genes from three different species, 88% of 42 randomly picked and sequenced genes were efficiently recombined. Furthermore, PTRec is a technically simple, fast, and reliable method that can be used for domain-and exon-shuffling or recombination of DNA fragments in general.

【 授权许可】

Unknown