摘要
尿酸氧化酶(urate oxidase,UOX EC1.7.3.3)是生物体内嘌呤代谢过程中的关键酶,能够催化尿酸转变为尿囊素。除人类和一些高等灵长目动物外,绝大多数生物体内能产生具有生物活性的尿酸氧化酶。这可能是因为进化而产生的优势,因为尿酸是强有力的氧自由基去除剂,所以人体内氧自由基的含量很低,因而因为衰老而产生癌症的几率大大下降。但由于先天或后天的原因引起的尿酸在体内生成增加或排泄障碍,就会导致高尿酸血症,从而引起各种相关的疾病,如痛风、肾结石等。此外,细胞的快速的裂解会引发以高尿酸血症为主要临床体征的肿瘤溶解综合症(tumor lysis syndrome, TLS)。利用UOX治疗TLS是国际公认的标准方法,同时逐渐成为治疗难治性痛风等高尿酸相关疾病的潜在治疗方法。
黄曲霉尿酸氧化酶(Aspergillus flavus urate oxidase;A. flavus UOX)是T-折叠家族蛋白质中的一员,该家族的蛋白质虽然序列一致性很低,但其结构却又很大的相似性,它们均表现出相似的寡聚化模式。X-ray衍射分析表明有生物活性的A. flavus UOX是由四个单体亚基(每个亚基含301个氨基酸,大小约34kDa)构成的同四聚体(135kDa),呈中空的隧道形。A. flavus UOX的氨基酸序列中含有三个半胱氨酸Cys35,Cys103,Cys290。通过与其他来源的UOX进行序列对比发现,三个半胱氨酸均非保守半胱氨酸,且不形成亚基内部和亚基之间的二硫键。但是有研究表明,三个半胱氨酸均有可能因为其硫原子的取向、被氧化或被连接一个加合物而影响尿酸氧化酶的活性。
为分析半胱氨酸残基对该酶的生物学活性的作用,选择利用大肠杆菌系统表达突变体C35A, C103A, C290A, C35A/C103A, C35A/C290A, C103A/C290A, C35A/C103A/C290A,将相应位点的半胱氨酸突变为丙氨酸。首先采用融合PCR的方法,构建了突变体的基因,将PCR产物克隆到T载体上进行核苷酸序列分析,通过Vector NTI与原始序列进行比对,确定获得了预期的突变体基因。将突变体基因进行双酶切后回收,亚克隆到表达载体pET-42a(+)的相应酶切位点,构建突变体的相应表达质粒。表达质粒进行双酶切鉴定,符合预期后,转化大肠杆菌BL21(DE3)感受态,经过氨苄青霉素筛选,即获得表达突变体的菌株。随机挑取突变体的菌株接种到LB培养基中进行培养,待菌密度OD600达到大约1.0时,加入终浓度为0.6mM的IPTG进行诱导,37℃诱导5小时。取诱导后菌液进行SDS-PAGE分析。结果显示,突变体蛋白均获得高效可溶性表达,大小约34kDa,符合预期,目的蛋白占蛋白总量的30%-55%。
根据前期的研究基础和突变体蛋白的性质,设计并优化了纯化工艺。首先采用phenyl sephrose FF疏水柱,捕获目的蛋白;接着采用DEAE阴离子柱纯化进行精细纯化,去除杂蛋白,大大降低内素素,蛋白纯度>90%;最后采用phenyl sephrose HP进行浓缩并进一步去除杂蛋白,获得纯度大于95%的突变体。
获得纯化的蛋白后,对其进行各种理化性质分析。SDS-PAGE显示与国外上市的同类制品具有一致的电泳结果。制备国外上市制品Rasburicase的兔多抗,并进行WB检测,显示突变体能够与兔多抗特异结合。在非还原状态下进行SDS-PAGE分析时,经由二硫键连接形成的异二聚体的量占蛋白总量的比例为0%-29%,经分析证明,Cys290对二聚体形成起的作用最大,Cys35最小。但HPLC结果显示,各突变体与原型均与国外上市标准品具有一致的保留时间,提示纯化后的产品以四聚体存在,而无可检测得到的二聚体存在。造成这种差异的原因为进行SDS-PAGE时,四聚体在SDS的作用下解聚,各亚基多肽链伸展后造成其中的Cys不同程度的暴露,而后在加热的作用下,互相靠近的不同亚基因其半胱氨酸被氧化形成二硫键而连接形成二聚体。这种现象提示我们在进行多亚基蛋白和含Cys的蛋白的研究和相关药物报批时,不能单独以SDS-PAGE结果作为其纯度等指标的检测手段。
体外活性方面,在还原条件下,各突变体活性为原型的41.8%-134.1%,其中C103A最高(134.1%),C35A/C103A/C290A最低(41.8%),造成这一现象的原因猜测为不同位点的Cys的存在对维系四级结构及活性位点的构象起到不同的作用;与氧化条件下相比,各突变体及原型在还原条件下活性均有不同的变化,增加最多的为C290A(增加约64.4%),降低最多的为C35A/C103A/C290A(降低15.5%),分析其原因为还原条件下,原型及突变体中氧化的Cys被还原,其上附着的加合物被去除的原因;最接近实际生产条件的氧化条件下,影响突变体活性的因素可视为以上两种情况的叠加,其中C103A活性增加最多(增加56.1%),C35A/C103A/C290A减少最多(减少27.1%)。综合分析,各个Cys虽然会因被氧化而影响酶的活性,但是对酶的四级结构的稳定和活性位点构象的维系可能会起到间接的作用。三个位点对活性的影响是累加的、复合的作用。将三个位点全部去除,酶的活性会遭到很大的破坏。但仅突变掉Cys103,能够使酶的体外活性得到很大的提高,这对该酶的体外应用(例如作为尿酸检测试剂)来说,意义重大。选择C103A, C290A,及C35A/C103A/C290A进行体内活性分析,结果表明:当三个位点均被突变掉后其活性有显著的下降。与体外活性结果相符。但是突变掉单一位点的Cys对UOX的体内活性影响不大,分析可能与体内特殊的氧化还原环境有关。
Urate oxidase (uricase; EC 1.7.3.3; or UOX) belongs to the purine degradation pathway and catalyzes in the presence of molecular oxygen the hydroxylation of uric acid into a metabolic product identified as the 5-hydroxyisourate(5-HIU). Urate oxidase is present in many species, but is absent in human and higher apes. This emphasizes an evolutionary advantage: it has been suggested that uric acid is a powerful antioxidant, so humans would have less free radicals and thus less cancer as a result of aging. However, congenital or acquired factors increased production of uric acid in the body or blocked excretion, can cause hyperuricemia, arising a variety of related diseases such as gout, kidney, etc. In addition, the rapid decomposition of cells can lead to hyperuricemia in the process of chemotherapy of tumors, resulting in tumor lysis syndrome (TLS). Uricase was thought of as the standard treatment for the TLS, and is becoming the potential, effective way for the hyperuriceamia.Use of urate oxidase treatment of these diseases is an effective way.
Aspergillus flavus urate oxidase is a member of T-fold family of proteins. The presently known T-fold proteins share a high structural similarity although they only exhibit a low amino acid sequence identity, they all showed a similar oligomerization pattern. X-ray diffraction analysis of the Aspergillus flavus urate oxidase showed that the asymmetric unit contains one monomer (34kDa) of 301 amino-acids and the whole homo-tetramer (135kDa) is built using the two-fold axes of the current I222 crystal symmetry. Each monomer is associated with one active site located at a dimmer interface. Three non-conservative cysteine amino acid (Cys35, Cys103, Cys290) present in the Aspergillus flavus urate oxidase sequence and they do not form a subunit disulfide bonds within and between subunits. However, studies have shown that sulfur atoms of three cystines are likely to affect the activity of urate oxidase because of their orientation, oxidation, or connection to the adducts.
To investigate the function of the cystine residues in the enzyme, mutants of the C35A, C103A, C290A, C35A/C103A, C35A/C290A, C103A/C290A, C35A/C103A/ C290A were expressed in E.coil expression system. First, the mutant gene was constructed by fusion PCR method. The PCR products were cloned into pMD-T vector for nucleotide sequence analysis. The obtained sequences were compared with the original sequence by Vector NTI software to verify the expected mutant gene. Then, the mutant gene was digested by Nde I/Hind III,extracted and subcloned into the corresponding restriction sites of the expression vector pET 42a (+) to construct the mutant expression plasmid respectively. Finally, expression plasmid was transformed into the E.coli BL21 (DE3) competent cells to obtain engineered strains by ampicillin selection on the LB plate. Randomly selected mutant strains were inoculated into LB culture medium until the OD600 reached about 0.6-1.0, and then 0.6mM of IPTG was added for induction for 5h at 37℃. The induced cells were analyzed by SDS-PAGE. The results showed that the mutant with expected size were high-level expressed successfully in a complete soluble form. The productions of the mutant amount to 30-50% of the total bacterial soluble proteins.
Based on the previous study and considering the characterizations of the mutant, the purified mutants were prepared. First, the mutant proteins were captured by Phenyl sepharose FF. Next, the mutants were further purified by DEAE sepharose FF to reduce host cell protein, nucleic acid and endotoxin. At last, the mutants were polished and reconcentrated by Phenyl sephrose HP. After three steps purification, the mutants were purified up to 95% purity.
The physical and chemical characterizations of the purified mutants were further evaluated. SDS-PAGE showed the mutants were similar to the commercial product Rasburicase. Western blot showed that the mutants can specifically react to the rabbit anti -Rauburicase serum. Non-reduced SDS-PAGE showed that dimmer of the mutants amount to 0%-29%,respectively. HPLC showed that the mutants performed the same reserve time to the original protein. From the HPLC result,it is induced that the mutants existed in the tetramer form under the normal circumstance. The controversial results between the SDS-PAGE and HPLC can be explained by the process of SDS-PAGE. In the process of the SDS-PAGE, the tetramer was disassociated, cystine residues were exposed, neighbouring cystine residues were oxidized into disulfide bond and further developed dimmer.
The bioactivity analysis in vitro was done under the reduced condition, the mutants showed 41.8%-134.1% activity compared to the original urate oxidase. Amount o f which, the mutant C103A performed highest acivity with 134.1%, the mutant C35A/C103A/C290A performed the lowest activity of 41.8%. The difference between the mutants and wild type urate oxidase can be explained by the different sites cystines residues have different action on the maintenance of the active centre conformation. Whereas in the reduced condition, the bioactivity of the mutants and wild-type urate oxidase changed obviously, amount of which the mutant C290A enhance up to 64.4%, the mutant C35A/C103A/C290A reduce up to 15.5. The reason can be related to the removal of the adduct connected to the cystine residues. In the process of the large-scale preparation, the bioactivity was influenced by the above reasons. Given the above results, the cystine residues have indirect action on the maintenance of the active centre conformation though the bioactivity of the urate oxidase was reduced because of oxidation of the cystines residues. The action of the three cystines was connected each other. If all the three cystines were removed, the bioactivity would reduce greatly. But if the Cys103 was replaced with ala alone, the bioactivity of the urate oxidase improved highly. The activity analysis in vivo was further evaluated on the mice hyperuricemia model. The result showed that the mutant C35A/C103A/C290A showed lowest activity of reducing uric acid, the mutants with single mutation showed similar activity to the wild type urate oxidase.
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