A potential method for one-step purification and direct immobilization of target protein in cell lysate with magnetic microbeads

Abstract

Functional carrier capable of covalently binding proteins from crude lysate under mild condition has become increasingly important in bioprocess. In this paper, a magnetic microbead that can covalently immobilize specific proteins directly from crude protein solution was developed, and its application conditions and stability were evaluated. The SpyCatcher was firstly modified with N-terminal cysteine by genetic methods and then the cys-SpyCatcher was covalently immobilized on epoxy-modified magnetic beads by epoxy-thiol reaction. The reaction between the epoxy and the only terminal thiol group in cys-SpyCatcher ensured the same orientation of the cys-SpyCatcher on the particle surface, while the maximum binding capacity of cys-SpyCatcher reached 23.6 μg/mg under the condition of 250 mM Na2SO4, pH 8.5 at initial cys-SpyCatcher concentration of 3 mg/mL. The functional magnetic microsphere (MS@cys-SpyCatcher) showed covalent site-specific capture property for enhanced green fluorescent protein fused with SpyTag (SpyTag-EGFP). A maximum binding capacity of (25 μg SpyTag-EGFP)/(mg beads) was far more than that of the previous carrier with SpyCatcher. In addition, the MS@cys-SpyCatcher could specifically capture the SpyTagged protein directly from the cell lysate, and this carrier showed good stability at pH 4–9 and temperature of 30–70ºC. Therefore, as a universal covalent capture carrier, it is believed that the MS@cys-SpyCatcher is useful in one-step purification and site-specific immobilization of the SpyTagged protein in bioprocess or biomedical application.