Vector construction and biotransformation of double subunits salvianolic acid- esterase
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摘要: 为了实现丹酚酸酯酶的外源性表达,利用了大肠杆菌和毕赤酵母两种表达系统,原核大肠杆菌采用p ET 28a和p ET 32a构建表达载体,并对重组大肠杆菌进行分别诱导表达和共表达;真核毕赤酵母采用p PIC9K构建共表达载体,对重组毕赤酵母进行诱导表达。研究结果表明,两种体系均能诱导表达出丹酚酸酯酶,蛋白在大肠杆菌系统中得到了高效表达,但未显示出该酶活力。毕赤酵母系统可使蛋白分泌性表达,表达产物具有一定的丹酚酸酯酶活力,但表达量不高。说明大肠杆菌系统体系更为适合大量表达丹酚酸酯酶,由此提供了一种该酶的外源表达方法。Abstract: In order to achieve the salvianolic acid- esterase express in vitro,the salvianolic acid- esterase were heterologous expressed by E.coli and Pichia pasioris respectively.The E.coil system constructed expression vector with p ET 28 a and p ET 32 a and carried out induced expression and coexpression of the recombinant E.coil. The Pichia pastoris system constructed coexpression vector with p PIC9 K and carried out induced expression of the recombinant Pichia pastoris. The research found that the salvianolic acid- esterase were induced in E.coli and Pichia pasioris.The protein was highly expressed in E.coli system,but it did not exhibit the enzyme activity. The salvianolic acid- esterase were expressed in Pichia pasioris,and the expression product had salvianolic acid-esterase activity.But the quantity of expression was not high.It showed that the E.coli expression system was more suitable for the salvianolic acid- esterase and provided a exogenous expression method of salvianolic acid-esterase.
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Keywords:
- salvianolic acid-esterase /
- E.coil /
- Pichia pastoris /
- cloning and expression
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[1] Chao C,Yin Y,Duan J,et al.Neuroprotective effect and underlying mechanism of sodium danshensu[3-(3,4-dihydroxyphenyl)lactic acid from Radix and Rhizoma Salviae miltiorrhizae=Danshen]against cerebral ischemia and reperfusion injury in rats[J].Phytomedicine,2015,22(2):283-289.
[2] Zhao G R,Zhang H M,Ye T X,et al.Characterization of the radical scavenging and antioxidant activities of danshensu and salvianolic acid B[J].Food and Chemical Toxicology,2008,46(1):73-81.
[3] 陈瑞战,张守勤,张永宏,等.超高压提取丹参素的研究[J].农业工程学报,2008,24(1):291-295. [4] 邹晓军,高小利.丹参中丹参素和原儿茶醛的微波提取工艺研究[J].中药材,2007,30(6):735-737. [5] 王金军,林琳,李景令,等.丹酚酸B水解生产丹参素的工艺优化[J].天然产物研究与开发,2015,27:715-719. [6] 关丹,高建梅,王何,等.丹参中水溶性成分提取新方法的研究[J].大连轻工业学院学报,2007,26(3):193-195. [7] 于翀,王莹,鱼红闪,等.丹酚酸B酶产物的分离[J].大连工业大学学报,2009,28(3):161-164. [8] 马明飞,李树金,金凤燮,等.人参皂苷葡萄糖苷酶基因在大肠杆菌中的表达及包涵体的变复性[J].大连工业大学学报,2012,31(1):8-11. [9] Laemmli U K.Cleavage of structural proteins during the assemble of the head of bacteriophage T4[J].Nature,1970,227:680-685.
[10] 刘俊梅,胡耀辉.薄层层析在多元醇检测中的应用[J].食品科技,2007,6:224-226. [11] Simone,Vincenzi,Andrea,et al.Anomalous electrophoretic behavior of a chitinase isoform from grape berries and wine in glycol chitin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels[J].Electrophoresis,2005,26(1):60-63.
[12] Richards J P,Bchinger H P,Goodman R H,et al.Analysis of the structural properties of c AMP-responsive element-binding protein(CREB)and phosphorylated CREB[J].Journal of Biological Chemistry,1996,271(23):13716-13723.
[13] Lucía,Vivian,Antonio,et al.Anomalous electrophoretic behavior of a very acidic protein:ribonuclease U2[J].Electrophoresis,2005,26:3407-3413.
[14] Armstrong D J,Roman A.The anomalous electrophoretic behavior of the human papillomavirus type 16 E7 protein is due to the high content of acidic amino acid residues[J].Biochemical and biophysical research communications,1993,192(3):1380-1387.
[15] 侯颖.rhdd ADAM15抗肿瘤及抗血管新生作用研究[D].无锡:江南大学,2014. [16] Yang W,Zhang L,Lu Z,et al.A new method for protein coexpression in Escherichia coli using two incompatible plasmids[J].Protein expression and purification,2001,22(3):472-478.
[17] Grosjean H,Fiers W.Preferential condon usage in prokaryotic genes:the optimal condon-anticondon interaction energy and the selective condon usage in efficiently expression genes[J].Gene,1982,18(3):199-209.
[18] 李单单.糖苷水解酶GH12家族活性架构中关键氨基酸的功能分析[D].威海:山东大学,2013. [19] Large T H,Rauh J J,De Mello,et al.Two molecular weight forms of muscarinic acetylcholine receptors in the avian central nervous system:switch in predominant form during differentiation of synapses[J].Proceedings of the National Academy of Sciences of the United States of America,1985,82(24):8785-8789.
[20] Schlegl R,Iberer G,Machold C,et al.Continuous matrixassisted refolding of protein[J].Journal of Chromatography A,2003,1009(1-2):119-132.
[21] Vallejo L F,Rinas U.Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins[J].Microbial Cell Factories,2004,3(1):85-100.
[22] Jungbauer A,Kaar W,Schlegl R.Folding and refolding of proteins in chromatographic beds[J].Current Opinion in Biotechnology,2004,15(5):487-494.
[23] Fidler A E,Lin J S,Chie WN,et al.Production of biologically active tethered ovine FSH beta alpha by the methylotrophic yeast Pichia pastoris[J].Mol Endocrinol,2003,30(2):213-225.
[24] Sreekrishna K,Nelles L,Potenz R,et al.High-level expression,purification,and characterization of recombinant human tumornecrosis factor synthesized in the methy lotrophic yeast Pichia pastoris[J].Biochem istry,1989,28(9):4117-4125.
[25] Baneyx F.Recombinant protein expression in Escherichia coli[J].Current opinion in biotechnology,1999,10(5):411-421.
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