(571aa) Protein Dissection and Shuffling for Generating Recombined Proteins | AIChE

(571aa) Protein Dissection and Shuffling for Generating Recombined Proteins


Chen, Y. - Presenter, Tsinghua University
Chen, T. - Presenter, Tsinghua University
Hua, H. - Presenter, Tsinghua University
Lin, Z. - Presenter, School of Medicine
Li, S. - Presenter, South China University of Technology

A simple method that allows for the facile recombination of distantly related proteins at multiple discrete sites was developed. The approach relies on protein random dissection to search for possible breakage points, which involves the use of a folding reporter to identify soluble shorter peptides independently of protein function. These dissection points are then used as candidate sites for protein recombination. To evaluate this method, two variants of aminoglycoside-3'-phosphotransferase (APHI and APHII) were chosen as model proteins, which share 31.4% amino acid identity. APH I and APH II were recombined at three sites, the library of 16 (24) chimeras was screened, among which 4/16 were found to show resistance to kanamycin, albeit at lower levels compared with the wild-type APH. We further applied random mutation to improve the activity of these chimeras. The best mutant showed a MIC of 800 µg/ml, which was an improvement of about 8-fold after one round of evolution. An interesting chimera which had a large extra insertion of about 100 amino acids into APH I at the first recombination site also showed a high catalytic efficiency with a MIC of 800 µg/ml. These results demonstrated that the protein shuffling method should be useful for creating artificial biocatalysts from different templates with low homology.