代谢型谷氨酸受体4胞内段的原核表达及纯化
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摘要
目的原核表达代谢型谷氨酸受体4胞内段(metabolic glutamate receptor 4 intracellular,GRM4 intracellular,GRM4 intra),并用Ni柱进行纯化。方法采用PAS(PCR-based accurate synthesis)法合成GRM4 intra基因序列,将目的片段和质粒p Czn1双酶切后,经连接,构建重组质粒pCzn1-GRM4 intra;将重组质粒转化至Rosseta菌株,IPTG诱导GRM4 intra蛋白的表达,表达的蛋白利用Ni柱亲和层析法纯化。结果经测序和双酶切鉴定证明质粒pCzn1-GRM4 intra构建正确;在IPTG诱导下,质粒能够在Rosseta菌株中高效表达,表达的蛋白相对分子质量约18 400,主要以可溶性性形式存在,纯度> 90%。结论成功获得了原核表达的GRM4 intra蛋白,为利用体外pull down方法鉴定GRM4的相互作用蛋白提供了参考。
Objective To express metabolic glutamate receptor 4 intracellular(GRM4 intra) in prokaryotic cells and purify the expressed product by nickel ion affinity chromatography. Methods GRM4 intra gene was synthesized by PCRbased accurate synthesis method. The target gene and plasmid p Czn1 were digested with NdeⅠand XbaⅠand linked,and the constructed recombinant plasmid pCzn1-GRM4 intra was transformed to E. coli Rosseta and induced with IPTG. The expressed protein was purified by nickel ion affinity chromatography. Results Recombinant plasmid pCzn1-GRM4 was constructed correctly as proved by restriction analysis and sequencing,and was highly expressed in E. coli Rosseta. The expressed protein,with a relative molecular mass of about 18 400,mainly existed in a soluble form,of which the purity reached more than 90%. Conclusion GRM4 intra was successfully expressed in prokaryotic cells,which provided a reference for identification of GRM4 interectome in pull down assays in vitro.
Objective To express metabolic glutamate receptor 4 intracellular(GRM4 intra) in prokaryotic cells and purify the expressed product by nickel ion affinity chromatography. Methods GRM4 intra gene was synthesized by PCRbased accurate synthesis method. The target gene and plasmid p Czn1 were digested with NdeⅠand XbaⅠand linked,and the constructed recombinant plasmid pCzn1-GRM4 intra was transformed to E. coli Rosseta and induced with IPTG. The expressed protein was purified by nickel ion affinity chromatography. Results Recombinant plasmid pCzn1-GRM4 was constructed correctly as proved by restriction analysis and sequencing,and was highly expressed in E. coli Rosseta. The expressed protein,with a relative molecular mass of about 18 400,mainly existed in a soluble form,of which the purity reached more than 90%. Conclusion GRM4 intra was successfully expressed in prokaryotic cells,which provided a reference for identification of GRM4 interectome in pull down assays in vitro.
引文
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[8] IACOVELLI L,ARCELLA A,BATTAGLIA G,et al. Pharmacological activation of m Glu4 metabotropic glutamate receptors inhibits the growth of medulloblastomas[J]. J Neurosci,2006,26(32):8388-8397.
[9] STEPULAK A,LUKSCH H,GEBHARDT C,et al. Expression of glutamate receptor subunits in human cancers[J]. Histochem Cell Biol,2009,132(4):435-445.
[10] SAVAGE S A,MIRABELLO L,WANG Z,et al. Genome-wide association study identifies two susceptibility loci for osteosarcoma[J]. Nat Genet,2013,45(7):799-803.
[11] YANG W,MAOLIN H,JINMIN Z,et al. High expression of metabotropic glutamate receptor 4:correlation with clinicopathologic characteristics and prognosis of osteosarcoma[J]. J Cancer Res Clin Oncol,2014,140(3):419-426.
[12] SHARP P M,LI W H. Codon usage in regulatory genes in Escherichia coli does not reflect selection for'rare'codons[J].Nucleic Acids Res,1986,14(19):7737-7749.
[13] SHARP P M,TUOHY T M,MOSURSKI K R. Codon usage in yeast:cluster analysis clearly differentiates highly and lowly expressed genes[J]. Nucleic Acids Res, 1986, 14(13):5125-5143.
[14] XIA H F,ZHANG X,JIN X H,et al. Preparation of Ni affinity chromatographic adsorbent and its application on purification of His-tagged protein[J]. J Jiangnan Univ(Nat Sci Ed),2010,9(6):685-689.(in Chinese)夏海锋,张显,金雄华,等.镍离子亲和层析介质的制备及其用于组氨酸标记蛋白质的纯化[J].江南大学学报(自然科学版),2010,9(6):685-689.
[15] JIANG H,ZHENG X F,LUO Y,et al. Preparation of Ni2+affinity gel and purification of His6-tag recombinant proteins[J].Bull Acad Mil Med Sci,2003,27(1):47-48,53.(in Chinese)江红,郑晓飞,罗瑛,等. Ni2+亲和胶的制备及其对带His6标签融合蛋白的纯化[J].军事医学科学院院刊,2003(1):47-48,53.
[2] XIAO B,CHEN L D,SUN C H,et al. Eukaryotic expression of m GluR4 and its role in the proliferation of breast cancer cell[J]. Lett Biotechnol,2017,28(2):109-113.(in Chinese)肖斌,陈丽丹,孙朝晖,等.代谢型谷氨酸受体4的真核表达及其在乳腺癌细胞增殖中的作用[J].生物技术通讯,2017,28(2):109-113.
[3] GRAY A L,HYDE T M,DEEP-SOBOSLAY A,et al. Sex differences in glutamate receptor gene expression in major depression and suicide[J]. Mol Psychiatry,2015,20(9):1057-1068.
[4] LI J,MENG H,CAO W,et al. MiR-335 is involved in major depression disorder and antidepressant treatment through targeting GRM4[J]. Neurosci Lett,2015,606(10):167-172.
[5] CHEN R,WANG H,JIANG W. Suppressive effect of metabotropic glutamate receptor 4 on neuropathic pain by Rab5[J]. J Shanghai Jiao Tong Univ(Med Sci),2014,34(2):139-143.(in Chinese)陈锐,王华,江伟.代谢型谷氨酸受体4通过Rab5抑制神经病理性痛[J].上海交通大学学报(医学版),2014,34(2):139-143.
[6] ZHENG B,ZHOU L M,ZHAO Q W,et al. Expression of metabotropic glutamate receptor 4 in cardiomyocytes differentiated from mouse embryonic stem cells in vitro[J]. J Zhejiang Univ(Med Sci),2012,41(4):366-372.(in Chinese)郑蓓,周立民,赵青威,等.代谢型谷氨酸受体4在ES细胞体外分化为心肌细胞中的表达研究[J].浙江大学学报(医学版),2012,41(4):366-372.
[7] WU Y L,DING H G. Role of metabotropic glutamate receptors in cancer development and progression[J]. Chin J Hepatol,2014,22(7):558-560.(in Chinese)武永乐,丁惠国.代谢型谷氨酸受体在肿瘤发生和发展中的作用[J].中华肝脏病杂志,2014,22(7):558-560.
[8] IACOVELLI L,ARCELLA A,BATTAGLIA G,et al. Pharmacological activation of m Glu4 metabotropic glutamate receptors inhibits the growth of medulloblastomas[J]. J Neurosci,2006,26(32):8388-8397.
[9] STEPULAK A,LUKSCH H,GEBHARDT C,et al. Expression of glutamate receptor subunits in human cancers[J]. Histochem Cell Biol,2009,132(4):435-445.
[10] SAVAGE S A,MIRABELLO L,WANG Z,et al. Genome-wide association study identifies two susceptibility loci for osteosarcoma[J]. Nat Genet,2013,45(7):799-803.
[11] YANG W,MAOLIN H,JINMIN Z,et al. High expression of metabotropic glutamate receptor 4:correlation with clinicopathologic characteristics and prognosis of osteosarcoma[J]. J Cancer Res Clin Oncol,2014,140(3):419-426.
[12] SHARP P M,LI W H. Codon usage in regulatory genes in Escherichia coli does not reflect selection for'rare'codons[J].Nucleic Acids Res,1986,14(19):7737-7749.
[13] SHARP P M,TUOHY T M,MOSURSKI K R. Codon usage in yeast:cluster analysis clearly differentiates highly and lowly expressed genes[J]. Nucleic Acids Res, 1986, 14(13):5125-5143.
[14] XIA H F,ZHANG X,JIN X H,et al. Preparation of Ni affinity chromatographic adsorbent and its application on purification of His-tagged protein[J]. J Jiangnan Univ(Nat Sci Ed),2010,9(6):685-689.(in Chinese)夏海锋,张显,金雄华,等.镍离子亲和层析介质的制备及其用于组氨酸标记蛋白质的纯化[J].江南大学学报(自然科学版),2010,9(6):685-689.
[15] JIANG H,ZHENG X F,LUO Y,et al. Preparation of Ni2+affinity gel and purification of His6-tag recombinant proteins[J].Bull Acad Mil Med Sci,2003,27(1):47-48,53.(in Chinese)江红,郑晓飞,罗瑛,等. Ni2+亲和胶的制备及其对带His6标签融合蛋白的纯化[J].军事医学科学院院刊,2003(1):47-48,53.