家蚕碱性磷酸酶原核表达纯化、结构与活性分析

doi:10.3864/j.issn.0578-1752.2017.14.019

摘要/Abstract

摘要： 【目的】碱性磷酸酶(alkaline phosphatase, ALP)是生物体内磷酸代谢的关键调控酶。不同物种ALP的性质与其生理功能密切相关。研究家蚕（Bombyx mori）ALP(BmALP)的性质和结构，为揭示ALP在昆虫体内的生理功能和调控机制提供依据。【方法】取5龄3 d家蚕中肠组织，利用Trizol法提取总RNA，然后以其为模板反转录合成cDNA。以家蚕中肠cDNA为模板，利用Primer Premier 6.0软件分别设计上下游引物，通过PCR克隆BmALP。将BmALP与不同的表达载体分别进行双酶切，然后连接并转化表达菌株，利用大肠杆菌表达重组蛋白。比较不同表达载体在上清中的表达情况，选择可溶性重组蛋白表达最好的载体，利用Origami B (DE3)细胞大量表达重组蛋白，借助Ni-NTA亲和层析纯化重组的His-Trx-BmALP蛋白，然后加入Prescission蛋白酶在4℃酶切20 h，再次利用Ni-NTA亲和层析去除融合标签His-Trx。利用凝胶过滤层析分析BmALP分子量及其在溶液中的状态，利用圆二色光谱研究BmALP的二级结构及其随温度的变化。通过酶活分析研究BmALP的最适pH、最适温度、Km、结构稳定性及金属离子对其酶学活性的影响。【结果】从家蚕中肠组织提取了总RNA，反转录合成了cDNA，并以该cDNA为模板成功克隆了BmALP。分别构建了BmALP的pSKB2、ppSUMO和pET32M.3C 3种表达载体。表达分析发现，pET32M.3C载体有助于重组的融合蛋白His-Trx-BmALP以可溶性蛋白的形式在细胞裂解后的上清液中表达，然后利用pET32M.3C载体大量表达重组蛋白，并通过Ni-NTA亲和层析纯化获得了可溶性的His-Trx-BmALP。加入Prescission蛋白酶酶切，再次通过Ni-NTA亲和层析去除了融合标签His-Trx。凝胶过滤分析显示BmALP在溶液中形成稳定的二聚体结构。圆二色光谱研究表明BmALP包含有α-螺旋结构，其含量随温度的升高逐渐减少。酶活分析揭示BmALP的最适pH和最适温度分别为 11.0和45℃。测定BmALP的Km值为1.40 mmol·L^-1。在10℃处理2 h后，BmALP的残余活性最高；35℃处理2 h后，其活性完全丧失。Mg²⁺和Zn²⁺促进了BmALP催化反应的进行，其最适浓度分别为40和5 mmol·L^-1。在20 mmol·L^-1浓度范围内，Cu²⁺激活了BmALP活性，其中10 mmol·L^-1时激活效果最好，浓度超过20 mmol·L^-1时，Cu²⁺则抑制了BmALP活性。【结论】克隆了家蚕碱性磷酸酶基因BmALP，表达纯化了BmALP蛋白并分析了其结构和性质，为深入研究其结构和功能打下了基础。

关键词: 家蚕, 碱性磷酸酶, 表达纯化, 结构, 性质

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 of B. 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 supernatant 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.

Key words: Bombyx mori, alkaline phosphatase, expression and purification, structure, property

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.

HE HuaWei, WANG YeJing, HOU Li, LI Yu, WEI ShuGuang, ZHAO Peng, JIANG WenChao, ZHAO Ping. Expression, Purification, Structure and Activity Analysis of Alkaline Phosphatase of Bombyx mori[J]. Scientia Agricultura Sinica, 2017, 50(14): 2837-2850.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.14.019

https://www.chinaagrisci.com/CN/Y2017/V50/I14/2837

参考文献

[1] Nakamura T. The phosphorus metabolism during the growth of the animal. The behavior of various phosphates and phosphoric acid compounds of Bombyx mori L.during growth. Mitt Med Akad Kioto, 1940, 28: 335-416.

[2] Bianchi U. Homologous alkaline phosphatases and homologous loci in two sibling species of European anopheline mosquitoes. Nature, 1968, 217(5126): 382-383.

[3] Verhaert P, Walgraeve H, Downer R. Alkaline phosphatase activity in the brain of the American cockroach Periplaneta americana L. The Histochemical journal, 1990, 22(11): 628-635.

[4] Yang M Y, Wang Z, Macpherson M, Dow J A, Kaiser K. A novel Drosophila alkaline phosphatase specific to the ellipsoid body of the adult brain and the lower Malpighian (renal) tubule. Genetics, 2000, 154(1): 285-297.

[5] Funk C J. Alkaline phosphatase activity in whitefly salivary glands and saliva. Archives of insect biochemistry and physiology, 2001, 46(4): 165-174.

[6] Sridhara S, Bhat J V. Alkaline and acid phosphatases of the silkworm, Bombyx mori L. Journal of Insect Physiology, 1963, 9(5): 693-701.

[7] Eguchi M, Sawaki M, Suzuki Y. Multiple forms of midgut alkaline phosphatase in the silkworm: new band formation and the relationship between the midgut and digestive fluid enzymes. Insect Biochemistry, 1972, 2(7): 297-304.

[8] Eguchi M, Sawaki M, Suzuki Y. Multiple forms of midgut alkaline phosphatase in the silkworm: separation and comparison of two isoenzymes. Insect Biochemistry, 1972, 2(6): 167-174.

[9] Eguchi M. Alkaline phosphatase isozymes in the alimentary canal of the silkworm//Isozymes I. Molecular Structure. New York: Academic Press, 1975: 315-332.

[10] Eguchi M, Yamashita Y. Genetic study on alkaline phosphatases of the midgut tissue and digestive fluid of the silkworm, Bombyx mori L. Journal of Sericultural Science of Japan, 1977, 46(6): 515-520. (in Japanese)

[11] Eguchi M, Daimon H, Kuriyama K. High alkalinity and function of proteases of digestive juice from the silkworm, Bombyx mori. Journal of Sericultural Science of Japan, 1986, 55(1): 46-53. (in Japanese)

[12] Eguchi M, Azuma M, Yamamoto H, Takeda S. Genetically defined membrane-bound and soluble alkaline phosphatases of the silkworm: their discrete localization and properties. Progress in clinical and biological research, 1990, 344: 267-287.

[13] Okada N, Azuma M, Eguchi M. Alkaline phosphatase isozymes in the midgut of silkworm: purification of high pH-stable microvillus and labile cytosolic enzymes. Journal of Comparative Physiology B, 1989, 159(2): 123-130.

[14] Eguchi M. Alkaline phosphatase isozymes in insects and comparison with mammalian enzyme. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1995, 111(2): 151-162.

[15] Waterhouse D. Functional differentiation of the hindgut epithelium of the blowfly larva into longitudinal bands. Australian Journal of Biological Sciences, 1955, 8(4): 514-529.

[16] Waterhouse D, Stay B. Functional differentiation in the midgut epithelium of blowfly larvae as revealed by histochemical tests. Australian Journal of Biological Sciences, 1955, 8(2): 253-277.

[17] Beadle D. The localisation of alkaline phosphatase in the midgut epithelium of Carausius morosus. Histochemie, 1971, 27(4): 370-372.

[18] Dimitriadis V, Kastritsis C. Ultrastructural analysis of the midgut of Drosophila auraria larvae-Distribution of alkaline phosphatase, acid phosphatase, leucine aminopeptidase, and glycogen. Cytologia, 1985, 50(4): 689-700.

[19] Jimenez D R, Gilliam M. Ultrastructure of the ventriculus of the honey bee, Apis mellifera (L.): cytochemical localization of acid phosphatase, alkaline phosphatase, and nonspecific esterase. Cell and Tissue Research, 1990, 261(3): 431-443.

[20] Yi S X, Adams T. Age- and diapause-related acid and alkaline phosphatase activities in the intestine and malpighian tubules of the Colorado potato beetle, Leptinotarsa decemlineata (Say). Archives of insect biochemistry and physiology, 2001, 46(3): 152-163.

[21] Mcnall R J, Adang M J. Identification of novel Bacillus thuringiensis Cry1Ac binding proteins in Manduca sexta midgut through proteomic analysis. Insect biochemistry and molecular biology, 2003, 33(10): 999-1010.

[22] Jurat-Fuentes J L, Adang M J. Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae. European Journal of Biochemistry, 2004, 271(15): 3127-3135.

[23] Cook B j, Nelson D R, Hipps P. Esterases and phosphatases in the gastric secretion of the cockroach, Periplaneta americana. Journal of Insect Physiology, 1969, 15(4): 581-589.

[24] Schmidt J O, Blum M S, Overal W L. Comparative enzymology of venoms from stinging Hymenoptera. Toxicon, 1986, 24(9): 907-921.

[25] 李文楚. 软化病感染家蚕的碱性磷酸酶活力测定及病理学研究. 华南农业大学学报, 2004, 25(4): 120-122.

LI W C. Studies on the activities of alkaline phosphatase and pathology of Bombyx mori infected with flacherie. Journal of South China Agricultural University, 2004, 25(4): 120-122. (in Chinese)

[26] Tsugikazu K, Yoshikatsu S, Takamitsu S. Multiple forms of human intestinal alkaline phosphatase: chemical and enzymatic properties, and circulating clearances of the fast-and slow-moving enzymes. Clinica Chimica Acta, 1981, 117(2): 167-187.

[27] Chang W S, Zachow K R, Bentley D. Expression of epithelial alkaline phosphatase in segmentally iterated bands during grasshopper limb morphogenesis. Development, 1993, 118(2): 651-663.

[28] Fernandez L E, Aimanova K G, Gill S S, Bravo A, Soberón M. A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae. Biochemical Journal, 2006, 394(1): 77-84.

[29] Yamamoto H, Azuma M, Eguchi M. Further characterization of alkaline phosphatase isozymes in the silkworm midgut: effects of amino acids and metal ions and comparison of sugar chains. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1991, 99(2): 437-443.

[30] 赵欣平, 舒畅, 杨芳, 刘克武, 喻东. 金属离子和脲对白蜡虫碱性磷酸酶的影响. 昆虫学报, 2002, 45(3): 318-322.

ZHAO X P, SHU C, Yang F, LIU K W, YU D. Effects of metal ions and urea on alkaline phosphatase from Ericerus pela (Chavannes). Acta Entomologica Sinica, 2002, 45(3): 318-322. (in Chinese)

[31] 赵欣平, 张久源, 杨守忠, 刘克武, 喻东. 白蜡虫碱性磷酸酶功能基团的研究. 昆虫学报, 2001, 44(3): 257-262.

ZHAO X P, ZHANG J Y, Yang S Z, LIU K W, YU D. Functional groups of alkaline phosphatase from Ericerus pela. Acta Entomologica Sinica, 2001, 44(3): 257-262. (in Chinese)

[32] 严盈, 刘万学, 万方浩. B型烟粉虱与温室白粉虱不同虫态的碱性磷酸酶性质比较. 昆虫学报, 2008, 51(1): 1-8.

YAN Y, LIU W X, WAN F H. Comparison of alkaline phosphatase in Bemisia tabaci B-biotype (Homoptera: Aleyrodidae) and Trialeurodes vaporariorum (Homoptera: Aleyrodidae) at different developmental stages. Acta Entomologica Sinica, 2008, 51(1): 1-8. (in Chinese)

[33] Ashrafi S H, Naqvi S, Qadri M. Alkaline phosphatase in the digestive system of the desert locust, Schistocerca gregaria (Forskal). 1969, 69(3): 183-191.

[34] Houk E J, Hardy J L. Alkaline phosphatases of the mosquito, Culex tarsalis Coquillett. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, 1984, 78(2): 303-310.

[35] Rauschenbach I Y, Bogomolova E, Gruntenko N, Adonyeva N, Chentsova N. Effects of juvenile hormone and 20-hydroxyecdysone on alkaline phosphatase activity in Drosophila under normal and heat stress conditions. Journal of Insect Physiology, 2007, 53(6): 587-591.

[36] Bourtzis K, Marmaras V J, Zacharopoulou A. Biochemical and genetic studies on alkaline phosphatase of Ceratitis capitata. Biochemical genetics, 1993, 31(9/10): 409-424.

[37] Miao Y G. Studies on the activity of the alkaline phosphatase in the midgut of infected silkworm, Bombyx mori L. Journal of Applied Entomology, 2002, 126(2/3): 138-142.

[38] 缪云根. 家蚕中肠碱性磷酸酶活性变化研究. 蚕业科学, 1988, 14(3): 154-158.

MIAO Y G. Study on alkaline phosphatase activity in midgut of silkworm, Bombyx mori. Science of Sericulture, 1988, 14(3): 154-158. (in Chinese)

[39] Itoh M, Kanamori Y, Takao M, Eguchi M. Cloning of soluble alkaline phosphatase cDNA and molecular basis of the polymorphic nature in alkaline phosphatase isozymes of Bombyx mori midgut. Insect biochemistry and molecular biology, 1999, 29(2): 121-129.

[40] Itoh M, Inoue T, Kanamori Y, Nishida S, Yamaguchi M. Tandem duplication of alkaline phosphatase genes and polymorphism in the intergenic sequence in Bombyx mori. Molecular Genetics and Genomics, 2003, 270(2): 114-120.

[41] 李长春, 罗英, 鲁成, 唐云明. 家蚕碱性磷酸酶的分离纯化与部分性质. 西南师范大学学报(自然科学版), 2005, 30(5): 930-934.

LI C C, LUO Y, LU C, TANG Y M. Isolation, purification and some properties of alkaline phosphatase from Bombyx mori. Journal of Southwest China Normal University (Natural Science),2005, 30(5): 930-934. (in Chinese)

[42] 何华伟, 位曙光, 王叶菁, 刘莉娜, 李珍珍, 赵朋, 常怀普, 赵萍. 家蚕bHLH转录因子Bmsage可溶性表达、纯化与结构分析. 生物工程学报, 2016, 32(10): 1395-1407.

HE H W, WEI S G, WANG Y J, LIU L N, LI Z Z, ZHAO P, CHANG H P, ZHAO P. Soluble expression, purification and structural analysis of the bHLH transcription factor Bmsage of Bombyx mori. Chinese Journal of Biotechnology, 2016, 32(10): 1395-1407. (in Chinese)

[43] Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 2016, 33(7): 1870-1874.

[44] 王叶菁, 刘莉娜, 高春雁, 李珍珍, 常怀普, 赵萍, 何华伟. 家蚕丝腺因子SGF-1的基因克隆及序列结构和表达特征与亚细胞定位. 蚕业科学, 2016, 42(2): 219-227.

WANG Y J, LIU L N, GAO C Y, LI Z Z, CHANG H P, ZHAO P, HE H W. Gene cloning, sequence structure, expression pattern and subcelluar localization of silk gland factor SGF-1 of Bombyx mori. Science of Sericulture, 2016, 42(2): 219-227. (in Chinese)

[45] 赵朋, 王叶菁, 位曙光, 刘莉娜, 李珍珍, 赵萍, 何华伟. 家蚕bHLH转录因子Bmdimm与Bmchip的相互作用. 中国农业科学, 2016, 49(10): 2027-2038.

ZHAO P, WANG Y J, WEI S G, LIU L N, LI Z Z, ZHAO P, HE H W. Interaction of bHLH transcription factor Bmdimm and Bmchip in Bombyx mori. Scientia Agricultura Sinica, 2016, 49(10): 2027-2038. (in Chinese)

[46] Wang Y, He H, Liu L, Gao C, Xu S, Zhao P, Xia Q. Inactivation and unfolding of protein tyrosine phosphatase from Thermus thermophilus HB27 during urea and guanidine hydrochloride denaturation. PloS one, 2014, 9(9): e107932.

[47] Liu L, Wang Y, Li Y, Lin Y, Hou Y, Zhang Y, Wei S, Zhao P, Zhao P, He H. LBD1 of vitellogenin receptor specifically binds to the female-specific storage protein SP1 via LBR1 and LBR3. PloS one, 2016, 11(9): e0162317.

[48] 廖金花, 陈清西. 金属离子对鲍鱼碱性磷酸酶活力的影响. 厦门大学学报(自然科学版), 2004, 43(增刊): 12-15.

LIAO J H, CHEN Q X. Effect of metal ions on the activity of alkaline phosphatase from Haliotis diversicolor. Journal of Xiamen University (Natural Science), 2004, 43(Suppl.): 12-15. (in Chinese)