Proline‐rich chaperones are compared computationally and experimentally for their abilities to facilitate recombinant butyrylcholinesterase tetramerization in CHO cells
Human butyrylcholinesterase (BChE), predominantly tetramers with a residence time of days, offers the potential to scavenge organophosphorus pesticides and chemical warfare agents. Efficient assembly of human BChE into tetramers requires an association with proline‐rich peptide chaperones. In this study, the incorporation of different proline‐rich peptide chaperones into BChE was investigated computationally and experimentally. First, we applied molecular dynamic (MD) simulations to interpret the interactions between proline‐rich chaperones with human BChE tetramer domains. The P24 chaperone which contains 24 prolines, promoted the association of BChE tetramer with a 74 % simulated helicity of BChE subunits, whereas the control without chaperone and BChE with an 8‐proline chaperone (P8) complex exhibited 55.8 % and 60.6 % predicted helicity, respectively. The interaction of proline‐rich chaperones with BChE subunits (B‐P) provides a conduit to facilitate the interactions between BChE subunits (B‐B) of the complex, which is mainly attributed to hydrophobic interactions and hydrogen‐bond binding. Experimental assessment of these two proline‐rich chaperones plus a 14‐proline chaperone (P14) was performed and confirmed that P24 has superior capability to facilitate recombinant BChE (rBChE) tetramerization with >60 % rBChE tetramer in P24‐transfected rBChE cells, whereas P14‐ and P8‐transfected rBChE cells have 44 % and 33 % rBChE tetramer respectively. The rBChE control had 14% tetramer. Finally, we developed a stable rBChE tetramer expression system in CHO cells by enriching P24 expression in rBChE expressing cells. Overall, our simulations provided a design concept for identifying proline‐rich peptides that promote the rBChE tetramerization in CHO cells.
