[Separation and purification of recombinant Nippostrongylus brasiliensis acetylcholinesterase from culture medium of genetic engineering Pichia pastoris].
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.
Zhang
Yaodong
Y
Department of Chemical Engineering, Xi'an Jiaotong University, Xi'an 710049, China.
Yang
Bolun
B
D000110:Acetylcholinesterase / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D003470:Culture Media / Q000737:chemistry*; D009559:Nippostrongylus / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*
D003470:Culture Media; D000110:Acetylcholinesterase