[Preparation of streptavidin mutant gel columns and purification of biotinylated protein]

Objective To make a site-directed mutation of streptavidin (SA) to reduce its affinity to biotin, to prepare a streptavidin mutant agarose gel column, and to find the optimal elution conditions to purify the biotin-modified protein. Methods The streptavidin mutant protein expressed in prokaryotes was purified using Ni-NTA resin, and the purified SA mutant protein was coupled with CNBr-activated BestaroseTM 4FF agarose gel to prepare the column. The biotin-modified protein was incubated with the gel column, and was eluted and recovered using NaOH at various concentrations. The purity of protein was detected by Western blotting and Coomassie brilliant blue staining. Results Five SA mutants were expressed and purified, and gel columns were successfully prepared. The results of ELISA showed that the affinity of SA-mutant to biotin-modified protein was lower than that of wild type SA. By purifying the biotin-modified protein with different gel columns, we screened out the SA mutant M2 gel column that could purify the biotin-modified protein with 10 mmol/L NaOH. And the M2 gel column could be used to re-purify biotin-modified protein. Conclusion The SA mutant agarose gel column has been successfully prepared, which provides a feasible and reliable option for the purification of biotin-modified protein.