Regulation of protein activity by conjugation to polymers is important in bioengineering and biomedical applications. The stability of protein-polymer conjugates may be improved by minimizing the number of polymer chains in the conjugate, as well as by choosing the optimal site on the protein for conjugation. In this work site-directed mutagenesis was used to prepare two pyrophosphatase (PPase) mutants with reactive thiol groups near to and far from the active center, respectively. Poly (2-hydroxyethyl methacrylate) (pHEMA) and poly(2-(dimethylamino) ethyl methacrylate) (pDMAEMA) were modified by introducing a pyridyl disulfide group at the polymer chain end, which was further reacted with the thiol group on the protein surface. Conjugation of pHEMA near the active center resulted in a clear and reversible decrease (or complete loss) of protein activity due to a conformational change in the tertiary structure of the protein. In contrast, conjugation of pDMAEMA to a specific site on the PPase surface far from the active center led to a significant increase in activity and stability at acidic pH because of the prevention of protein aggregation. In summary, our results provided a general approach for efficient on-demand control of protein activity and stability based on site-specific conjugation of polymers.<br/>This work was financially supported by the National Science Fund for Distinguished Young Scholars (21125418) and the National Natural Science Foundation of China (21334004, 21474071).

Regulation of protein properties via site-specific polymer conjugation

249th National Meeting (2015)

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