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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2017, Vol. 37 Issue (11): 74-82    DOI: 10.13523/j.cb.20171110
研究报告     
季也蒙毕赤酵母菌尿酸酶基因的克隆、重组表达及表征
饶菁菁, 景一娴, 邹明月, 胡小蕾, 廖飞, 杨晓兰
临床检验诊断学教育部重点实验室 重庆医科大学检验医学院 重庆 400016
Clone, Expression and Characterization of the Uricase from Meyerozyma guilliermondii
RAO Jing-jing, JING Yi-xian, ZOU Ming-yue, HU Xiao-lei, LIAO Fei, YANG Xiao-lan
Key Laboratory of Clinical Laboratory Diagnostics of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China
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摘要： 目的：克隆一种季也蒙毕赤酵母菌的尿酸酶蛋白编码基因，并通过重组表达和表征进行确认。方法：测定酵母细胞株C.G.M.C.C 2.1008的rRNA序列鉴定其所属种，串联质谱分析此天然尿酸酶肽段序列搜索同源蛋白，测定转录组验证其诱导表达属性，据季也蒙毕赤酵母ATCC 6260基因组信息推断所得尿酸酶（Uniprot id：A5DFP1）的编码序列设计引物，从C.G.M.C.C 2.1008细胞株cDNA中PCR扩增尿酸酶基因，测序后再克隆至定向表达载体pDE1及pDE2中构建带6His标签的重组表达质粒pDE1-MGU和pDE2-MGU，同时构建无6His标签的表达质粒R-MGU。在大肠杆菌BL21（DE3）中诱导表达此真菌尿酸酶，SDS-PAGE和MALDI-TOF-MS测定肽段分子质量，以尿酸为底物测定其动力学参数及抑制剂敏感性等性质，并与天然尿酸酶进行比较。结果：rRNA序列表明此菌株为季也蒙毕赤酵母，串联质谱分析胰蛋白酶解肽段表明此天然真菌尿酸酶与尿酸酶A5DFP1高度相似，转录组测序支持此尿酸酶基因高效诱导表达。用所设计引物经PCR从cDNA快速获得编码序列，T载体测序显示其与A5DFP1编码序列仅在第435位碱基不同但氨基酸仍相同。SDS-PAGE发现重组R-MGU肽段约35kDa；MALDI-TOF-MS发现其肽段约17.43kDa且与天然酶一致，但按氨基酸序列计算分子质量仅为33.98kDa，表明其可能存在化学修饰。重组表达带6His标签尿酸酶纯化后比活性接近6.0U/mg；R-MGU米氏常数、代表抑制剂的抑制常数及分子质量与天然尿酸酶无差别，但R-MGU很难纯化，使其相同条件下的热稳定性比纯化天然酶略差。结论：成功克隆了一种季也蒙毕赤酵母菌的尿酸酶，并实现其活性形式的重组表达。
关键词： 重组表达;  季也蒙毕赤酵母尿酸酶;  基因克隆;  表征    
Abstract: Objective:To clone the uricase gene of the Meyerozyma guilliermondii(C.G.M.C.C 2.1008) for recombinant expression in Escherichia coli and further characterization. Methods:The strain was identified by sequences of 28S rRNA D1/D2 and ITS. Partial peptide sequences were recognized through trypsin digestion and MS/MS analyses. The induction expression of the uricase was verified by RNA-seq. The target gene was amplified by PCR through precise primers designed according to the coding sequence of a homologus putative uricase of Meyerozyma guilliermondii ATCC 6260. The cloned coding sequence was inserted into the expression vectors pDE1 and pDE2 to construct the plasmids pDE1-MGU and pDE2-MGU bearing 6His tags. After the deletion of 6His tag and the linking peptide, the vector for the non-tagged MGU was constructed and denoted R-MGU. The plasmids were transformed into E. coli BL21 (DE3) for induced expression. The recombinant protein was characterizedby SDS-PAGE, MALDI-TOF-MS and the related enzymatic properties. Result:The sequences of rRNA supported that it was a strain of Meyerozyma guilliermondii. MS/MS analyses supported its high homology to a putative uricase A5DFP1 deduced from the genomic sequence of Meyerozyma guilliermondii ATCC 6260 and its induced expression by uric acid was further verified by RNA-seq. After PCR cloning with a pair of precise primers, the coding sequence of the targeted MGU showed the only different base at the 435th site but gave the same amino acid residue. After recombinant expression, R-MGU had the peptide weight of about 35kDa by SDS-PAGE, but of 17.43 by MALDI-TOF-MS that was consistent with that of the wildtype, supporting the unidentified chemical modification of MGU. The maximum specific activity of the recombinant form of the uricase was about 6.0U/mg. Of R-MGU, the Km, Ki of representative inhibitors and molecular weight had no significantly differences from the wildtype. However, the thermostability of R-MGU was slightly worse than the wildtype, due primarily to the low purity of the sample. Conclusion:From Meyerozyma guilliermondii(C.G.M.C.C 2.1008), MGU gene was cloned and expressed successfully as the active enzyme in Escherichia coli.
Key words: Meyerozyma guilliermondii uricase    Gene cloning    Recombinant expression    Characterization
收稿日期: 2017-07-31 出版日期: 2017-11-15
ZTFLH:  Q781  
基金资助: 国家自然科学基金资助项目（201402108，31570862，81773625）
通讯作者: 杨晓兰     E-mail: xiaolanyang666@yeah.net
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引用本文:

饶菁菁, 景一娴, 邹明月, 胡小蕾, 廖飞, 杨晓兰. 季也蒙毕赤酵母菌尿酸酶基因的克隆、重组表达及表征[J]. 中国生物工程杂志, 2017, 37(11): 74-82.

RAO Jing-jing, JING Yi-xian, ZOU Ming-yue, HU Xiao-lei, LIAO Fei, YANG Xiao-lan. Clone, Expression and Characterization of the Uricase from Meyerozyma guilliermondii. China Biotechnology, 2017, 37(11): 74-82.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20171110    或    https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I11/74

[1] 武文明, 曾昭淳,李小彦,等. 产朊假丝酵母尿酸酶的纯化和特性研究. 西南大学学报(自然科学版), 2008, 30(3):84-89. Wu W M, Zeng Z C, Li X Y,et al. Purification and characterization of the uricase from Candida utilis.Journal of Southwest University (Natural Science Edition),2008, 30(3):84-89.
[2] Ishizaka N, Ishizaka Y, Toda E, et al. Association between serum uric acid, metabolic syndrome, and carotid athero-sclerosis in Japanese individuals. Arterioscler Thromb Vasc Biol, 2005, 25(5):1038-1044.
[3] Duncan P, Gochman N, Bayse D, et al. A candidate reference method for uric acid in serum. Ⅱ. Interlaboratory testing. Clinical Chemistry, 1982, 28(2):291-293.
[4] Koyama Y, Ichikawa T, Nakano E. Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding the Candida utilis Urate Oxidase (Uricase). Journal of Biochemistry, 1996, 120(5):969-973.
[5] Pui C H,Relling M V, Lascombes F, et al. Urate oxidase in prevention and treatment of hyperuricemia associated with lymphoid malignancies.Leukemia, 1997,11(11):1813-1816.
[6] 朱献军, 刘建国, 黎高翔.尿酸氧化酶基因的克隆、表达及其产物的应用.生物工程学报, 2001, 17(1):68-72. Zhu X J, Liu J G, Li G X.Cloning and expression of urate oxidase and its application in serum uric acid analysis.Chinese Journal of Biotechnology,2001, 17(1):68-72.
[7] Wu J, Yang X, Wang D, et al. A numerical approach for kinetic analysis of the nonexponential thermoinactivation process of uricase.Protein Journal, 2016, 35(4):1-12.
[8] 赵运胜,卜友泉,廖飞. 用简并引物快速克隆苛求芽孢杆菌尿酸酶的编码序列.重庆医科大学学报,2017,42(2):219-224. Zhao Y S, Bu Y Q,Liao F.Rapid cloning gene sequence of the uricase from Bacillus fastidious by degenerated primer. J Chongqing Med Univ,2017, 42(2):219-224.
[9] 赵运胜, 赵利娜, 杨根庆,等. 苛求芽孢杆菌胞外尿酸酶的表征及其在血清尿酸测定中的应用.华中科技大学学报(医学版), 2007,36(2):239-242. Zhao Y S, Zhao L N, Yang G Q,et al. Characterization of an extracellular uricase from Bacillus fastidious and its application in direct kinetic assay of serum uric acid. Acta Med Univ Sci Technol Huazhong,2007, 36(2):239-242.
[10] Liao F, Zhu X Y, Wang Y M, et al. The comparison of the estimation of enzyme kinetic parameters by fitting reaction curve to the integrated Michaelis-Menten rate equations of different predictor variables. Journal of Biochemical & Biophysical Methods, 2005, 62(1):13-24.
[11] Liu X, Wen M, Li J, et al. High-yield expression, purification, characterization, and structure determination of tag-free Candida utilis uricase. Applied Microbiology & Biotechnology, 2011, 92(3):529-537.
[12] 吴双林, 陈斌, 刘成倩,等. 猪尿酸氧化酶在大肠杆菌中的表达、纯化与部分酶学性质分析.生物工程学报, 2009, 25(11):1664-1670. Wu S L, Chen B, Liu C Q, et al. Expression in Escherichia coli, purification and enzymatic properties of porcine urate oxidase. Chin J Biotech, 2009, 25(11):1664-1670.
[13] Feng J, Wang L, Liu H, et al. Crystal structure of Bacillus fastidious uricase reveals an unexpected folding of the C-terminus residues crucial for thermostability under physiological conditions. Applied Microbiology & Biotechnology, 2015, 99(19):7973-7986.
[14] 陈丹, 韩晶, 史洪娜,等. 球形节杆菌尿酸酶的表达、纯化及其活性.中国生物制品学杂志, 2014, 27(3):351-355. Chen D, Han J, Shi H N,et al. Expression, purification and activity of Arthrobacter globiformis uricase.Chinese Journal of Biologicals,2014, 27(3):351-355.
[15] Suzuki K, Sakasegawa S I, Misaki H, et al. Molecular cloning and expression of uricase gene from Arthrobacter globiformis in Escherichia coli and characterization of the gene product. Journal of Bioscience & Bioengineering, 2004, 98(3):153-158.
[16] Huang Y, Chen Y, Yang X, et al. Optimization of pH values to formulate the bireagent kit for serum uric acid assay. Biotechnology & Applied Biochemistry, 2015, 62(1):137.
[17] Zhang C, Yang X, Feng J, et al. Effects of modification of amino groups with poly(ethylene glycol) on a recombinant uricase from Bacillus fastidiosus. Biosci Biotechnol Biochem,2010,74(6):1298-1301.
[18] Zhang C, Yang X, Gao A, et al. Comparison of modification of a bacterial uricase with N-hydroxysuccinimide esters of succinate and carbonate of monomethoxyl poly(ethylene glycol). Biotechnol Appl Biochem, 2014,61(6):683-690.
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