Separation and Purification of Recombinant Nippostrongylus brasiliensis Acetylcholinesterase from Culture Medium of Genetic Engineering Pichia pastoris
强阴离子色谱法从毕赤酵母培养液中分离纯化重组巴西日圆线虫乙酰胆碱酯酶

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 x 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 66 000, 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 2 837 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.