中国农业科学2017，Vol.50Issue(14)：2837-2850,14.DOI:10.3864/j.issn.0578-1752.2017.14.019
家蚕碱性磷酸酶原核表达纯化、结构与活性分析
Expression, Purification, Structure and Activity Analysis of Alkaline Phosphatase of Bombyx mori
摘要
Abstract
[Objective] Alkaline phosphatase (ALP) is the key enzyme in the metabolism of phosphoric acid in vivo.The properties of ALP in different species are closely related to their physiological functions.The characterization of the property and structure of Bombyx mori ALP (BmALP) will facilitate to reveal the physiological function and regulation mechanism of ALP in insects.[Method] The total RNA was extracted by Trizol method from the midgut ofB.mori larvae on day 3 of the 5th instar,and then cDNA was synthesized with the extracted total RNA as the template by reverse transcription.The upstream and downstream primers were designed by Primer Premier 6.0 software,and BmALP was cloned with the synthesized cDNA as the template by PCR.BmALP and different expression vectors were double digested,respectively,then ligated and transformed into the expression strain.The recombinant protein was expressed by Escherichia coli.The expressions of different expression vectors in the supematant were compared,and the vector with the best expression of soluble recombinant protein was chosen.The recombinant protein was expressed in large scale using Origami B (DE3) cells,and digested with Prescission protease at 4℃ for 20 h followed by the purification via Ni-NTA affinity chromatography.Then the fusion His-Trx tag was removed using Ni-NTA affinity column again.The molecular weight and the state of BmALP in solution were analyzed by gel filtration chromatography.The secondary structure of BmALP and the effects of temperature on its structure were studied by circular dichroism spectroscopy.The optimum pH,optimum temperature,Km,structural stability and the effect of metal ions on the activity of BmALP were studied by the activity assay.[Result] The total RNA was extracted from the midgut of B.mori and cDNA was synthesized by reverse transcription.BmALP was successfully cloned with the cDNA as the template.The expression vectors of BmALP with pSKB2,ppSUMO and pET32M.3C were constructed,respectively.The expression analysis showed that the pET32M.3C vector facilitated the expression of the recombinant fusion protein His-Trx-BmALP in the form of a soluble protein in the supernatant of cell lysate.Then the recombinant BmALP was expressed in large scale with the pET32M.3C vector.The soluble recombinant His-Trx-BmALP was purified via Ni-NTA affinity chromatography.After the digestion of His-Trx-BmALP by Prescission protease,the fusion His-Trx tag was removed by Ni-NTA affinity column.Gel filtration analysis showed that BmALP formed a stable dimer in solution.Circular dichroism spectroscopy showed BmALP contained α-helical structure,and its content decreased with increasing temperature.Enzymatic activity analysis revealed that the optimum pH and temperature of BmALP were 11.0 and 45 ℃,respectively.The Km of BmALP was measured to be 1.40 mmol·L-1.After 2 h incubation at 10℃,BmALP had the highest residual activity,and the residual activity was completely lost after incubation at 35℃ for 2 h.Mg2+ and Zn2+ promoted the catalytic reaction of BmALP with the optimal concentration of 40 and 5 mmol·L-1,respectively.Cu2+ activated BmALP activity within 20 mmol·L-1,and the optimal concentration was 10 mmol·L-1,however,Cu2+ inhibited the activity of BmALP while its concentration was higher than 20 mmol·L-1.[Conclusion] BmALP was cloned.BmALP protein was expressed and purified and its structure and properties were analyzed.The results of this study provided a basis for further study of its structure and function.关键词
家蚕/碱性磷酸酶/表达纯化/结构/性质Key words
Bombyx mori/alkaline phosphatase/expression and purification/structure/property引用本文复制引用
何华伟,王叶菁,侯丽,李瑜,位曙光,赵朋,蒋文超,赵萍..家蚕碱性磷酸酶原核表达纯化、结构与活性分析[J].中国农业科学,2017,50(14):2837-2850,14.基金项目
国家自然科学基金(31402139,31572465)、国家自然科学基金重点项目(31530071)、重庆市基础科学与前沿技术研究专项(cstc2015jcyjA00040,cstc2015jcyjBX0035)、中央高校基本科研业务费(XDJK2013A019)、西南大学博士基金(SWU112111) （31402139,31572465）
