A facile method to oriented immobilization of His-tagged BirA on Co3+-NTA agarose beads

Publication date: Available online 18 September 2018Source: Enzyme and Microbial TechnologyAuthor(s): Xu Li, Rou Wang, Hanyu Cao, Xu Ting, Lulu Han, Chundong Huang, Lingyun JiaAbstractA facile and economical method was established for the oriented immobilization of biotin ligase (BirA) on Co3+-NTA sepharose through H2O2 oxidation of Co2+ and His-tag. His-tag of the BirA were designed at both N-terminal (His-BirA) and C-terminal (BirA-His), respectively. Immobilization of the His-BirA was performed, realized to 92.85% using by 10 mM H2O2 without compromising catalytic activity. Because amounts of ions on matrix were far more than that of the immobilized BirA, EDTA should be used to remove residual ions before catalyzing, while it should be limited to lower than 30 mM, and imidazole ranging from 50 to 250 mM could be added in the catalytic system. When 10 mM EDTA and 50 mM imidazole were used, over 90% of substrates were obtained from the matrix. Moreover, the His-BirA showed higher immobilization rate than the BirA-His, while both of them appeared high catalytic abilities at pH ranging from 6.5 to 9.0, indicating versatile options in the biotinylation of proteins with different pH stabilities. Under the best catalytic conditions, the both immobilized His-BirA and BirA-His exhibited the same activity as the free. When the enzyme was incubated at different pH (pH 3.0, 4.0, 5.0, 10.0 and 11.0) and temperature (40 °C, 50 °C and 60 °C), the immobilized His-BirA s...
Source: Enzyme and Microbial Technology - Category: Biotechnology Source Type: research