重组假丝酵母尿酸酶中聚合物的分析与评价

目的：分析重组假丝酵母尿酸酶中聚合物的形成原因及其对结构功能的影响。方法：通过分子排阻高效液相色谱（SEC-HPLC）结合多角度激光散射仪分离聚合物并测定其相对分子质量；将含有聚合物的蛋白进行还原及非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），确定聚合物的形成是否与二硫键有关；通过液质联用仪鉴定聚合物中存在的二硫键；通过测定圆二色谱评价聚合物的形成对蛋白二级结构的影响；通过多角度激光散射仪测定二硫键形成对蛋白稳定性的影响；通过酶活性测定评价聚合物形成对酶活性的影响。结果：重组假丝酵母尿酸酶正常以同源四聚体的形式存在，37℃条件下可通过链间二硫键形成更高级别的聚合物，高级聚合物的形成对蛋白二级结构及稳定性的影响不大，但会降低酶活性。结论：本文综合多种手段对尿酸酶中聚合物的成因及影响进行分析，测定结果可为该制品的后续研究及质量控制提供依据。

Objective: To analyze the cause of polymers in recombinant Candida uricase and its effect on structure and function. Methods: The polymers were separated by size exclusion-high performance liquid chromatography(SEC-HPLC) combined with multi-angle laser light scattering(MALLS) to measure their relative molecular weights. The proteins containing polymers were analyzed by reduced and non-reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) to determine whether the formation of polymers was related to disulfide bonds. The disulfide bonds in the polymers were identified by liquid chromatography-mass spectrometry (LC-MS);The influence of polymers formation on secondary structure of protein was evaluated by circular dichroism. The disulfide bonds' effect on thermal stability of protein was determined by MALLS. The enzyme activity was also measured to determine whether it was affected by polymerization. Results: The protein normally exists in the form of homologous tetramers,and higher level polymers can be formed gradually through inter-chain disulfide bonds at 37℃. The formation of higher level polymers has no significant effect on secondary structure and stability of protein,but it could reduce its enzyme activity. Conclusion: The causes and effects of polymers in uricase were analyzed by various methods,which can provide basis for the follow-up research and quality control of the products.

[1] Friedman TB,Polanco GE,Appold JC,et al.On the loss of uricolytic activity during primate evolution -.Silencing of urate oxidase in a hominoid ancestor[J].Comp Biochem Physiol B,1985,81(3):653
[2] Yeldandi AV,Yeldandi V,Kumar S,et al.Molecular evolution of the urate oxidase-encoding gene in hominoid primates:nonsense mutations[J].Gene,1991,109(2):281
[3] Richette P,Bardin T.Gout[J].Lancet,2010,375(9711):318
[4] Malaguarnera M,Vacante M,Russo C,et al.A single dose of rasburicase in elderly patients with hyperuricaemia reduces serum uric acid levels and improves renal function[J].Expert Opin Pharmacother,2009,10(5):737
[5] Schlesinger N,Yasothan U,Kirkpatrick P.Pegloticase[J].Nat Rev Drug Discov,2011,10(1):17
[6] Alakel N,Middeke JM,Schetelig J,et al.Prevention and treatment of tumor lysis syndrome,and the efficacy and role of rasburicase[J].Onco Targets Ther,2017,10:597
[7] Dave AJ,Kelly VM,Krishnan E.Pegloticase and the patient with treatment-failure gout[J].Expert Rev Clin Pharmacol,2012,5(5):501
[8] George RJ,Sundy JS.Pegloticase for treating refractory chronic gout[J].Drugs Today (Barc),2012,48(7):441
[9] Yood RA,Ottery FD,Irish W,et al.Effect of pegloticase on renal function in patients with chronic kidney disease:a post hoc subgroup analysis of 2 randomized,placebo-controlled,phase 3 clinical trials[J].BMC Res Notes,2014,7:54
[10] Colloc'H N,Girard E,Dhaussy AC,et al.High pressure macromolecular crystallography:the 140-MPa crystal structure at 2.3 A resolution of urate oxidase,a 135-kDa tetrameric assembly[J].Biochim Biophys Acta,2006,1764(3):391
[11] Kratzer JT,Lanaspa MA,Murphy MN,et al.Evolutionary history and metabolic insights of ancient mammalian uricases[J].Proc Natl Acad Sci U S A,2014,111(10):3763
[12] Roberts CJ.Non-native protein aggregation kinetics[J].Biotechnol Bioeng,2007,98(5):927
[13] Fathallah AM,Chiang M,Mishra A,et al.The effect of small oligomeric protein aggregates on the immunogenicity of intravenous and subcutaneous administered antibodies[J].J Pharm Sci,2015,104(11):3691
[14] Ahmadi M,Bryson CJ,Cloake EA,et al.Small amounts of sub-visible aggregates enhance the immunogenic potential of monoclonal antibody therapeutics[J].Pharm Res,2015,32(4):1383
[15] Moussa EM,Panchal JP,Moorthy BS,et al.Immunogenicity of therapeutic protein aggregates[J].J Pharm Sci,2016,105(2):417
[16] Liu X,Wen M,Li J,et al.High-yield expression,purification,characterization,and structure determination of tag-free Candida utilis uricase[J].Appl Microbiol Biotechnol,2011,92(3):529