Abstract:

To develop a simple, fast and highly efficient method for the separation and purification of recombinant Nippostrongylus brasiliensis acetylcholinesterase (NbAChE) from culture medium of genetic engineering Pichia pastoris, Q-Sepharose Fast Flow chromatographic medium was used. The chromatographic column was 20 cm×3.5 cm i.d. The elution buffer A was 20 mmol/L NaH2PO4-Na2HPO4 (pH 8) and buffer B was 1 mol/L NaCl+20 mmol/L NaH2PO4-Na2HPO4 (pH 8). The elution gradient was nonlinear. It was firstly eluted with 10%B for 300 min, then with 30%B for 300 min, finally with 100%B for 300 min. The flow rate of mobile phase was 6 mL/min. The obtained recombinant NbAChE was proved to be homogeneous on sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and its relative molecular mass was estimated to be approximately 66000, which was consistent with that reported in literature. The total activity recovery of this purification method was 52.6% and the purification factor was 3.87. The final specific activity of recombinant NbAChE was 2837 U/mg. This chromatographic process is simple and highly efficient. It can be used to separate and purify recombinant NbAChE from culture medium of Pichia pastoris harboring NbAChE gene.

Key words: acetylcholinesterase, culture medium, Pichia pastoris, purification , separation, anion exchange chromatography