Ogataea minuta来源的内切-β-N-乙酰氨基葡糖苷酶的异源表达和纯化

在大肠杆菌中表达有活性的Ogataea minuta来源的内切β-N-乙酰氨基葡糖苷酶（Endo-Om），利用pET系统过量表达Endo-Om。在优化宿主菌及培养条件后，镍柱纯化目的蛋白质，并检测纯化后的Endo-Om对荧光标志的寡糖链的水解活性。经IPTG诱导后，目的蛋白质可以很好地表达，但大部分存在包涵体中。通过培养条件优化，16 ℃在Overnight ExpressTM Instant LB 培养基（默克）中培养，实现了Endo-Om在大肠杆菌中的可溶性表达和纯化，并检测到了纯酶的水解活性。在大肠杆菌中成功纯化了有活性的Endo-Om蛋白，为加速研究该酶的结构和功能奠定了基础。
To express active endo-beta-N-acetylglucosaminidase（ENGase）from methylotrophic yeast Ogataea minuta（Endo-Om） in Escherichia coli. The Endo-Om was overexpressed by using pET system. After optimizing host strainand culture condition，we purified the recombinant protein by using Ni2+ metal chelating column.Using the purified Endo-Om，ENGase activity against fluorescent-labeled oligosaccharide was measured. The recombinant protein was well expressed by IPTG induction，however，the majority remained in the insoluble fraction. The insolubility of the recombinant was improved by culturing the E. coli cells in Overnight ExpressTM Instant LB Medium（Merck Millipore） at 16 ℃. The purified protein showed hydrolysis activity to the fluorescent- labeled oligosaccharide. The functional Endo-Om was successfully obtained in this study，it could accelerate the study on its structural and biological function