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1 生物工程学报 Chin J Biotech 2009, November 25; 25(11): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 工业生物技术 吴双林 1, 陈斌 2,3, 刘成倩 2, 欧瑜 1, 易建中 2 1, , , : 本研究报道了猪尿酸氧化酶 (Porcine urate oxidase, puox) 的原核表达载体的构建 puox 的蛋白表达条件的优化以及对 puox 经纯化后进行活性检测和酶学性质分析 利用 RT-PCR 从猪肝总 RNA 中克隆 puox, 定向插入原核表达载体 pet30a(+) 中, 构建表达载体 pet30a(+)/puox, 并转化到大肠杆菌 (Escherichia coli) BL21(DE3) 中 重组质粒 pet30a(+)/puox 经双酶切鉴定和序列分析, 证实已成功构建了重组表达载体 重组表达菌经 IPTG 诱导表达了约为 41 kd 的蛋白, 与预期分子量一致, 并对 puox 蛋白表达条件进行了优化, 表达的蛋白主要以包涵体的形式存在于细胞中, 包涵体经过变性 复性后, 用 Ni 2+ -NTA 对复性蛋白进行亲和纯化, 并对纯化蛋白进行了活性检测和酶学性质分析, 纯化的重组 puox 的比活为 IU/mg, 并发现重组蛋白在最佳温度 热稳定性等方面与天然 puox 相同, 为后续动物实验奠定重要的基础 : 猪尿酸氧化酶, 表达, 包涵体, 纯化, 生物活性 Expression in Escherichia coli, purification and enzymatic properties of porcine urate oxidase Shuanglin Wu 1, Bin Chen 2, 3, Chengqian Liu 2, Yu Ou 1, and Jianzhong Yi 2 1 School of Life Science and Technology, China Pharmaceutical University, Nanjing , China 2 Animal Husbandry and Veterinary Research Institute, Shanghai Academy of Agricultural Science, Shanghai , China 3 College of Veterinary Medicine, Nanjing Agricultural University, Nanjing , China Abstract: The aims of this research were to construct prokaryotic expression vector containing the gene of porcine urate oxidase(puox), optimize the conditions of the expression of puox in recombinant Escherichia coli BL21(DE3), and analyze the in vitro activity and the enzymological properties of puox. The puox gene was amplified by RT-PCR from the extracted total RNA of porcine liver, and was inserted into the prokaryotic expression vector pet30a(+) to construct a recombinant expression vector pet30a(+)/puox. We identified the recombinant vector by endonuclease digestion and sequence analysis. The puox gene was amplified and cloned into the vector pet30a(+) successfully. And then the recombinant vector was transformed into E. coli Received: July 22, 2009; Accepted: September 22, 2009 Supported by: Shanghai Pujiang Program (No. PJ[20]). Corresponding author: Yu Ou. ouyu1967@163.com Jianzhong Yi. yijianzhong@yahoo.com 上海市浦江人才计划 (No.PJ[20]) 资助

2 : 1665 BL21(DE3). The expression of puox with a molecular of approximately 41 kd was induced by IPTG. We also optimized the expression conditions of the recombinant protein. The recombinant protein was mostly located in the cytoplasm and it was insoluble. After the inclusion body was solved in 8 mol/l urea and refolding in 2 mol/l urea, the recombinant protein was collected and purified by Ni 2+ -NTA column. This recombinant protein had a specific activity of IU/mg and showed similar properties of optimum temperature and thermal stability, base on the enzymatic assay and analysis of enzymological properties. These results would help to analyze the in vivo activity by testing animal. Keywords: porcine urate oxidase, expression, inclusion body, purification, biological activity (Urate oxidase; Uricase, EC , UOX), CO 2 H 2 O [1-3] 2,, [4-5] (CGA/AGA TGA),,, [5-7],,, [8],,,, [9-11], [12-13], (PEG) [8,14-15],,, (Porcine urate oxidase, puox) 84%,,,, 1 材料与方法 1.1 材料 E. coli BL21, pet30a EcoR I Hind III T4 DNA (NEB ); dntps KOD (TaKaRa ); (Axygen ); RNA (BioTeke ) 1.2 方法 puox cdna, RNA RNA, 1%, RNA cdna, 20 o C puox RT-PCR GenBank (Accession No. M27697), Primer Premier 5.0 : 5 -GATCGAATTCATGGCTCATT ACCGTAATGAC-3 ( EcoR I ); : 5 -CTGGTAAGCTTTCACAGCCTTGA AGTCAGC-3 ( Hind III ) TGA puox cdna PCR puox PCR : 94 o C 3 min; 94 o C 30 s, 55 o C 30 s, 68 o C 70 s, 40 ; 68 o C 10 min PCR pet30a(+)/puox puox, EcoR I Hind III pet30a(+), PCR 16 o C, E. coli BL21, ;, PCR

3 1666 ISSN CN /Q Chin J Biotech November 25, 2009 Vol.25 No.11 EcoR I Hind III,, pet30a(+)/puox puox E. coli BL21 IPTG : (50 g/ml) LB, 37 o C 200 r/min, 1% (50 g/ml) LB, OD , ( mmol/l) IPTG, 37 o C 250 r/min,, 4 h,,, 10% SDS-PAGE, IPTG : IPTG, 22 o C 25 o C 28 o C 31 o C 34 o C 37 o C 3 h, SDS-PAGE, IPTG : IPTG,, h, SDS-PAGE, IPTG puox 1.2.4,, SDS-PAGE A(20 mmol/l Tris-HCl, 50 mmol/l NaCl, ph 8.0),, 0.5% Triton X-100 A, B(8 mol/l, 0.1 mol/l -, 20 mmol/l Tris-HCl, 50 mmol/l NaCl, ph 8.0) 2 h,, C(2 mol/l, 20 mmol/l Tris-HCl, 50 mmol/l NaCl, ph 8.0) 24 h, 4 o C, Ni 2+ -NTA, His Bind Resin 3 ml, 10 ml (10 mmol/l, 500 mmol/l NaCl, 20 mmol/l Tris-HCl), 10 ml (20 mmol/l, 500 mmol/l NaCl, 20 mmol/l Tris-HCl), 10 ml (250 mmol/l, 500 mmol/l NaCl, 20 mmol/l Tris-HCl), 1.5 ml puox [16] 3 ml 2.5 ml 0.1 mol/l ph %, 0.5 ml,,, 25 o C 293 nm : ph o C, 1 mol Bradford, BSA [17], 20 o C 30 o C 40 o C 50 o C 60 o C 70 o C 20 o C 30 o C 40 o C 50 o C 60 o C 70 o C min,, ph ph, ph 7.0~10.0 ph 3.0~12.0, 40 o C 40 min, ( ph 3.0~ mol/l - ; ph 7.0~ mol/l ; ph 8.0~ mol/l - ; ph 10.0~ mol/l - ) 2 结果 2.1 puox RT-PCR 扩增和重组质粒的鉴定 RNA, RT-PCR, 1000 bp, puox ( 1) pet30a(+)/puox EcoR I/Hind Ⅲ,

4 : bp ( 2), ; GenBank, 7 h 图 1 puox 基因的 RT-PCR 扩增 Fig. 1 Amplification of puox gene by RT-PCR. M: DNA marker DL2000; 1: RT-PCR product. 图 3 pet30a(+)/puox 重组蛋白 IPTG 的诱导浓度分析 Fig. 3 Effect of inducing concentration of IPTG on the expression of pet30a(+)/puox recombinant protein. M: protein molecular marker; 1: pet30a(+) induced with IPTG; 2: pet30a(+)/puox without induction; 3 8: pet30a(+)/puox induced with IPTG 0.05, 0.1, 0.2, 0.5, 1.0, 1.5 mmol/l respectively. 图 2 阳性克隆的 EcoR I/Hind III 双酶切鉴定 Fig. 2 Identification of positive clone by the double enzyme digestion of EcoR I/Hind III. M: DNA marker DL2000; 1: pet30a(+)plasmid; 2: the digested product of pet30a(+); 3: recombinant vector pet30a(+)/puox; 4: the digested product of pet30a(+)/puox; 5: RT-PCR product. 2.2 puox 在 E. coli BL21 中 IPTG 诱导表达条件的优化 IPTG ( mmol/l) 4 h, SDS-PAGE,, 41 kd,, IPTG 0.1 mmol/l, ( 3), IPTG 0.1 mmol/l 0.1 mmol/l IPTG, 22 o C 25 o C 28 o C 31 o C 34 o C 37 o C 3 h, SDS-PAGE, 31 o C, ( 4) 31 o C 0.1 mmol/l IPTG, 31 o C h, SDS-PAGE, 7 h, ( 5), 图 4 pet30a(+)/puox 重组蛋白的诱导温度的分析 Fig. 4 Effect of inducing temperature on the expression of pet30a(+)/puox recombinant protein. M: protein molecular weight marker; 1: pet30a(+) induced with IPTG; 2: pet30a(+)/puox without induction; 3 8: pet30a(+)/puox induced with 0.1 mmol/l IPTG at 22, 25, 28, 31, 34, 37 respectively. 图 5 pet30a(+)/puox 重组蛋白的诱导时间分析 Fig. 5 Effect of inducing time on expression of pet30a(+)/ puox recombinant protein. M: protein molecular weight marker; 1: pet30a(+) induced with IPTG; 2: pet30a(+)/puox without induction; 3-7: pet30a(+)/puox induced with 0.1 mmol/l IPTG at 31 for 1, 3, 5, 7, 10 h respectively.

5 1668 ISSN CN /Q Chin J Biotech November 25, 2009 Vol.25 No 目的蛋白可溶性分析和分离纯化, IPTG 0.1 mmol/l, 31 o C 7 h 6,, puox, 2.4 puox 酶活力的测定 puox : mg/ml; 2.81 IU/ml; : IU/mg 2.5 酶的最适温度和热稳定性 20 o C~70 o C 20 o C 40 o C, 40 o C 40 o C,, 70 o C 40 o C 67% 40 o C 20 o C~60 o C, 60 o C,, 70 o C 30 min, 7%, 80 o C 30 min ( 8) 图 6 重组蛋白 puox 可溶性分析和分离制备包涵体 Fig. 6 Identification of expression forms of recombinant protein puox and isolation of inclusion body. M: protein molecular weight marker; 1: total bacterial protein before induction; 2: total bacterial protein after induction; 3: preciption after altrasonication; 4: supernantant after altrasonication; 5: the first supernantant of washing pellet; 6: the second supernantant of washing pellet; 7: inclusion body., Ni 2+ -NTA SDS-PAGE, 41 kd ( 7) 图 8 温度对 puox 酶活的影响 Fig. 8 Effect of temperature on the enzyme activity of the recombinant protein puox. determination of the optimal temperature of the recombinant protein puox; test of thermal stability of the recombinant protein puox. 图 7 纯化蛋白 puox 的 SDS-PAGE 分析 Fig. 7 SDS-PAGE analysis of the purification of the protein puox. M: protein molecular weight marker; 1: inclusion body; 2: total protein in 8 mmol/l urea; 3: total protein after refolding; 4: flow-through fraction of Ni affinity column; 5: washing of Ni affinity column; 6 8: elution of Ni affinity column. 2.6 酶最适 ph 和 ph 稳定性 ph 9.5 ph 9.5, ph 9.5 ph 3.0~12.0 ph ph 5.0, 90% ph 3.0, 14%( 9)

6 : 1669 图 9 ph 对 puox 酶活的影响 Fig. 9 Effect of ph on the enzyme activity of the recombinant protein puox. determination of the optimal ph of the recombinant protein puox; test of ph stability of the recombinant protein puox. 3 讨论,,, (Tumor lysis syndrome, TLS) [18], TLS,, [19-21],, puox BL21(DE3), DNA,,,,, 16 o C, 25 o C,,, puox puox,,,, puox puox puox [22], puox ph 9.5, puox ph 8.4, ph puox puox REFERENCES [1] Hayashi S, Fujiwara S, Noguchi T. Degradation of uric acid in fish liver peroxisomes. Intraperoxisomal localization of hepatic allantoicase and purification of its peroxisomal membrane-bound form. J Biol Chem, 1989, 264(6): [2] Alamillo JM, Cárdenas J, Pineda M. Purification and molecular properties of urate oxidase from Chlamydomonas reinhardtii. Biochim Biophys Acta, 1991, 1076(2): [3] Bhargava AK, Lal H, Pundir CS. Discrete analysis of serum uric acid with immobilized uricase and peroxidase. J Biochem Biophys Methods, 1999, 39(3): [4] Yeldandi AV, Yeldandi V, Kumar S, et al. Molecular evolution of the urate oxidase-encoding gene in hominoid primates: nonsense mutations. Gene, 1991, 109(2): [5] Oda M, Satta Y, Takenaka O, et al. Loss of urate oxidase activity in hominoids and its evolutionary implications. Mol Biol Evol, 2002, 19(5): [6] Wu XW, Lee CC, Muzny DM, et al. Urate oxidase: primary structure and evolutionary implications. Proc Natl Acad Sci USA, 1989, 86(23): [7] Wu XW, Muzny DM, Lee CC, et al. Two independent mutational events in the loss of urate oxidase during hominoid evolution. J Mol Evol, 1992, 34(1): [8] Kelly SJ, Delnomdedieu M, Oliverio MI, et al. Diabetes insipidus in uricase-deficient mice: a model for evaluating therapy with poly(ethylene glycol)-modified uricase. J Am Soc Nephrol, 2001, 12(5): [9] Li J, Chen Z, Hou L, et al. High-level expression, purification, and characterization of non-tagged

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