| 초록 |
In this study, we have successfully combined the conventional site-directed mutation method with Gibson assembly procedures for simultaneous mutagenesis of multiple locations. A fluorescent protein mOrange is different from mStrawberry at four different amino acid residues. To convert the nucleotide sequence of mOrange into that of mStrawberry, we first designed the inversely paring primers for Quick-change mutagenesis of individual target sites of mOrange. Since the resulting five fragments have homologous ends, they could be subsequently joined by the Gibson assembly method to generate the gene of mStrawberry. As a results, we could convert mOrange into mStrawberry in a one-pot reaction, averting repeated steps of mutagenesis and cloning. Proposed strategy should be able to provide a facile route to engineering proteins with greater speed and flexibility. |
