肉鸡谷胱甘肽S-转移酶A3基因的克隆及蛋白的表达和纯化
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摘要
[目的]谷胱甘肽S-转移酶A3(GSTA3)是GSTs超家族中α家族的一员,具有解毒功能,同时还参与抗氧化应激引起的信号通路调节,另外在类固醇和前列腺素的合成中也必不可少。[方法]本试验采用RT-PCR方法扩增肉鸡GSTA3基因,连接pZeroBack/blunk克隆质粒,再与表达载体pET-28a连接,并转入E.coli BL21(DE3)中获得pET-28a-GSTA3重组表达质粒,使用IPTG诱导其蛋白的表达,应用镍柱亲和层析法纯化蛋白,并对其进行SDS-PAGE鉴定,使用谷胱甘肽-S转移酶活性测定试剂盒对重组蛋白进行活性检测。[结果]试验成功构建重组表达质粒pET-28a-GSTA3,并在大肠杆菌中成功表达,其大小为29.6 kDa,与预期一致,并且表达的重组GSTA3蛋白具有生物活性。[结论]本试验成功获得高纯度的可溶性GSTA3融合蛋白,为其在家禽领域的进一步研究奠定了基础。
[Objective] Glutathione S-transferase A3(GSTA3) is a major part of GST alpha subfamily, which possesses alexipharmic property. It is also involved in the regulation of signal pathways induced by oxidative stress and in the synthesis of steroids and prostaglandin. The proposed study was aimed to….. [Methods] The GSTA3 gene was amplified by RT-PCR method and the amplified ingredients were inserted into the pZeroBack/blunk cloning vector, and the product was connected to the pET-28 a. The construct was then transformed into E. coli BL21 to obtain recombinant expression plasmid pET-28 a-GSTA3. Protein expression was induced by IPTG. The GSTA3 fusion protein was purified by nickel column affinity chromatography, and the purified protein was identified by Western Blot and SDS-PAGE. The bioactivity of GSTs was examined using assay kit. [Results] The test successfully exhibited the recombinant expression vector pET-28 a-GSTA3. GSTA3 gene was successfully expressed in E. coli and the recombinant protein was around 29.6 kDa, and the expressed recombinant GSTA3 protein was biologically active. [Conclusion] This experiment successfully obtained high-purity soluble GSTA3 fusion protein, which established solid basis for further research in the field of poultry.
[Objective] Glutathione S-transferase A3(GSTA3) is a major part of GST alpha subfamily, which possesses alexipharmic property. It is also involved in the regulation of signal pathways induced by oxidative stress and in the synthesis of steroids and prostaglandin. The proposed study was aimed to….. [Methods] The GSTA3 gene was amplified by RT-PCR method and the amplified ingredients were inserted into the pZeroBack/blunk cloning vector, and the product was connected to the pET-28 a. The construct was then transformed into E. coli BL21 to obtain recombinant expression plasmid pET-28 a-GSTA3. Protein expression was induced by IPTG. The GSTA3 fusion protein was purified by nickel column affinity chromatography, and the purified protein was identified by Western Blot and SDS-PAGE. The bioactivity of GSTs was examined using assay kit. [Results] The test successfully exhibited the recombinant expression vector pET-28 a-GSTA3. GSTA3 gene was successfully expressed in E. coli and the recombinant protein was around 29.6 kDa, and the expressed recombinant GSTA3 protein was biologically active. [Conclusion] This experiment successfully obtained high-purity soluble GSTA3 fusion protein, which established solid basis for further research in the field of poultry.
引文
[1]Johansson A S, Mannervik B. Human glutathione transferase A3-3, a highly efficient catalyst of double-bond isomerization in the biosynthetic pathway of steroid hormones[J]. Journal of Biological Chemistry, 2001, 276(35):33061-33065.
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[3]Raffalli-Mathieu F, Orre C, Stridsberg M, et al. Targeting human glutathione transferase A3-3 attenuates progesterone production in human steroidogenic cells[J]. Biochemical Journal, 2008, 414(1):103-109.
[4]Omata Y, Saito Y, Fujita K, et al. Induction of adaptive response and enhancement of PC12 cell tolerance by lipopolysaccharide primarily through the upregulation of glutathione S-transferase A3 via Nrf2 activation[J]. Free Radical Biology and Medicine, 2008, 45(10):1437-1445.
[5]Buetler T M, Slone D, Eaton D L. Comparison of the aflatoxin B1-8,9-epoxide conjugating activities of two bacterially expressed alpha class glutathione S-transferase isozymes from mouse and rat[J]. Biochemical and Biophysical Research Communications, 1992, 188(2):597-603.
[6]Ilic Z, Crawford D, Vakharia D, et al. Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1[J]. Toxicology and Applied Pharmacology, 2010, 242(3):241-246.
[7]Berhane K, Widersten M, Engstr?m A, et al. Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases[J]. Proceedings of the National Academy of Sciences of the United States of America, 1994, 91(4):1480-1484.
[8]孟迅吾, 夏维波. 类固醇激素与骨[J]. 中国医学科学院学报, 2003, 25(3):237-239.
[9]刘爽, 胡宝成. 原核系统可溶性表达策略[J]. 生物技术通讯, 2005, 16(2):172-175.
[10]孙柏欣, 刘长远, 陈彦,等. 基因表达系统研究进展[J]. 现代农业科技, 2008(2):205-207.
[11]解庭波. 大肠杆菌表达系统的研究进展[J]. 长江大学学报:自然科学版, 2008, 5(3):77-82.
[12]Nuc P, Nuc K. Recombinant protein production in Escherichia coli[J]. Postepy Biochemistry, 2006, 52(4):448-456.
[13]王智慧. 人谷胱甘肽硫转移酶Pi基因的克隆及其蛋白的表达和纯化[D].郑州:郑州大学,2004.
[14]Weickert M J, Pagratis M, Curry S R, et al. Stabilization of apoglobin by low temperature increases yield of soluble recombinant hemoglobin in Escherichia coli[J]. Applied and environmental microbiology, 1997, 63(11):4313-4320.
[15]Baneyx F. Recombinant protein expression in Escherichia coli[J]. Current Opinion in Biotechnology, 1999, 10(5):411-421.
[16]Yu G Q,Jiang S Q,Li Y L. Optimizing expression of recombinant 26-kilodalton glutathione S-transferases of Schistosoma japonicum in Escherchia coli[J]. Chinese Journal of Zoonoses, 2006, 22(8):770-773.
[17]Shaw M K, Ingraham J L. Synthesis of Macromolecules by Escherichia coli near the Minimal Temperature for Growth[J]. Journal of Bacteriology, 1967, 94(1):157-164.
[18]Hesse S, Jernstr?m B, Martinez M, et al. Inhibition of binding of benzo(a)pyrene metabolites to nuclear DNA by glutathione and glutathione S-transferase B[J]. Biochemical and biophysical research communications, 1980, 94(2):612-617.
[19]Nair R, Salvi P, Banerjee S, et al. Yeast extract mediated autoinduction of lacUV5 promoter: an insight[J]. New Biotechnology, 2009, 26(6):282-288.
[20]Fu X, Wei D, Tong W. Effect of yeast extract on the expression of thioredoxin-human parathyroid hormone from recombinant Escherichia coli[J]. Journal of Chemical Technology and Biotechnology Biotechnology, 2010, 81(12):1866-1871.
[21]Butt T R, Edavettal S C, Hall J P, et al. SUMO fusion technology for difficult-to-express proteins[J]. Protein Expression and Purification, 2005, 43(1):1-9.
[2]Johansson A S, Mannervik B. Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3[J]. Journal of Biological Chemistry, 2002, 277(19):16648-16654.
[3]Raffalli-Mathieu F, Orre C, Stridsberg M, et al. Targeting human glutathione transferase A3-3 attenuates progesterone production in human steroidogenic cells[J]. Biochemical Journal, 2008, 414(1):103-109.
[4]Omata Y, Saito Y, Fujita K, et al. Induction of adaptive response and enhancement of PC12 cell tolerance by lipopolysaccharide primarily through the upregulation of glutathione S-transferase A3 via Nrf2 activation[J]. Free Radical Biology and Medicine, 2008, 45(10):1437-1445.
[5]Buetler T M, Slone D, Eaton D L. Comparison of the aflatoxin B1-8,9-epoxide conjugating activities of two bacterially expressed alpha class glutathione S-transferase isozymes from mouse and rat[J]. Biochemical and Biophysical Research Communications, 1992, 188(2):597-603.
[6]Ilic Z, Crawford D, Vakharia D, et al. Glutathione-S-transferase A3 knockout mice are sensitive to acute cytotoxic and genotoxic effects of aflatoxin B1[J]. Toxicology and Applied Pharmacology, 2010, 242(3):241-246.
[7]Berhane K, Widersten M, Engstr?m A, et al. Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases[J]. Proceedings of the National Academy of Sciences of the United States of America, 1994, 91(4):1480-1484.
[8]孟迅吾, 夏维波. 类固醇激素与骨[J]. 中国医学科学院学报, 2003, 25(3):237-239.
[9]刘爽, 胡宝成. 原核系统可溶性表达策略[J]. 生物技术通讯, 2005, 16(2):172-175.
[10]孙柏欣, 刘长远, 陈彦,等. 基因表达系统研究进展[J]. 现代农业科技, 2008(2):205-207.
[11]解庭波. 大肠杆菌表达系统的研究进展[J]. 长江大学学报:自然科学版, 2008, 5(3):77-82.
[12]Nuc P, Nuc K. Recombinant protein production in Escherichia coli[J]. Postepy Biochemistry, 2006, 52(4):448-456.
[13]王智慧. 人谷胱甘肽硫转移酶Pi基因的克隆及其蛋白的表达和纯化[D].郑州:郑州大学,2004.
[14]Weickert M J, Pagratis M, Curry S R, et al. Stabilization of apoglobin by low temperature increases yield of soluble recombinant hemoglobin in Escherichia coli[J]. Applied and environmental microbiology, 1997, 63(11):4313-4320.
[15]Baneyx F. Recombinant protein expression in Escherichia coli[J]. Current Opinion in Biotechnology, 1999, 10(5):411-421.
[16]Yu G Q,Jiang S Q,Li Y L. Optimizing expression of recombinant 26-kilodalton glutathione S-transferases of Schistosoma japonicum in Escherchia coli[J]. Chinese Journal of Zoonoses, 2006, 22(8):770-773.
[17]Shaw M K, Ingraham J L. Synthesis of Macromolecules by Escherichia coli near the Minimal Temperature for Growth[J]. Journal of Bacteriology, 1967, 94(1):157-164.
[18]Hesse S, Jernstr?m B, Martinez M, et al. Inhibition of binding of benzo(a)pyrene metabolites to nuclear DNA by glutathione and glutathione S-transferase B[J]. Biochemical and biophysical research communications, 1980, 94(2):612-617.
[19]Nair R, Salvi P, Banerjee S, et al. Yeast extract mediated autoinduction of lacUV5 promoter: an insight[J]. New Biotechnology, 2009, 26(6):282-288.
[20]Fu X, Wei D, Tong W. Effect of yeast extract on the expression of thioredoxin-human parathyroid hormone from recombinant Escherichia coli[J]. Journal of Chemical Technology and Biotechnology Biotechnology, 2010, 81(12):1866-1871.
[21]Butt T R, Edavettal S C, Hall J P, et al. SUMO fusion technology for difficult-to-express proteins[J]. Protein Expression and Purification, 2005, 43(1):1-9.