Efficient purification of a recombinant tag-free thermostable Kluyveromyces marxianus uricase by pH-induced self-cleavage of intein and expression in Escherichia coli
Efficient purification of a recombinant tag-free thermostable Kluyveromyces marxianus uricase by pH-induced self-cleavage of intein and expression in Escherichia coli
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通过 pH 诱导的内含肽自裂解和在大肠杆菌中的表达，有效纯化重组无标签热稳定性马克斯克鲁维酵母尿酸酶。
DOI：
10.1007/s13205-018-1422-9
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发表时间：
2018-09
期刊：
影响因子：
2.8
通讯作者：
Hong Jiong
中科院分区：
文献类型：
--
作者：
Wang Bangchun;Luo Laipeng;Wang Dongmei;Ding Rui;Hong Jiong
尿酸酶作为一种重要的保健相关蛋白，广泛应用于肿瘤溶解综合征的治疗和血清尿酸诊断试剂盒的研制。本研究从耐热酵母Kluyveromycesmarxianus中克隆了一种新的耐热尿酸酶基因（KmUOX）。将尿酸酶与自切割内含肽和纤维素结合亲和标签融合，并在大肠杆菌BL 21（DE 3）中表达。通过与廉价纤维素的结合和pH变化诱导的内含肽原位切割，通过一步柱纯化策略有效地纯化了无标签尿酸酶（KmUOX），产率为77.11%。这种无标签尿酸酶的Km、Vmax和Kcat值分别为67.60 µM、56.35 µM/（min mg）和32.74 S−1。该酶具有较好的热稳定性，在40 °C孵育90 h后仍保持79.75%的活性。因此，这种pH诱导的可自切割内含肽系统与用于亲和层析的纤维素基质相结合，被证明是一种有效且低成本的重组尿酸酶纯化方法。此外，KmUOX的稳定性使其可用于临床应用。
Uricase as an important healthcare-related protein is extensively used in the treatment of tumor lysis syndrome and in the manufacture of serum uric-acid diagnostic kits. In this study, a gene of a new thermostable uricase (KmUOX) was cloned from thermotolerant yeastKluyveromyces marxianus. The uricase was fused with a self-cleaving intein and cellulose-binding affinity tag and expressed inEscherichia coliBL21 (DE3). Through the binding to inexpensive cellulose and in situ intein cleavage induced by a pH change, tag-free uricase (KmUOX) was efficiently purified with a 77.11% yield via a single-step column purification strategy. This tag-free uricase showedKm,Vmax, andKcatvalues of 67.60 µM, 56.35 µM/(min mg), and 32.74 S−1, respectively. Furthermore, this pure uricase was relatively thermostable and retained 79.75% of activity when incubated at 40 °C for 90 h. Thus, this pH-induced self-cleavable intein system combined with a cellulose matrix for affinity chromatography is proven here to be an effective and low-cost method for recombinant-uricase purification. Moreover, the stability of KmUOX makes it useful for clinical applications.
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