Arthur Patrick K, Alvarado Luigi J, Dayie T Kwaku
Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20742-3360, USA.
Protein Expr Purif. 2011 Apr;76(2):229-37. doi: 10.1016/j.pep.2010.11.008. Epub 2010 Nov 24.
RNAs, more than ever before, are increasingly viewed as biomolecules of the future, in the versatility of their functions and intricate three-dimensional folding. To effectively study them by nuclear magnetic resonance (NMR) spectroscopy, structural biologists need to tackle two critical challenges of spectral overcrowding and fast signal decay for large RNAs. Stable-isotope nucleotide labeling is one attractive solution to the overlap problem. Hence, developing effective methods for nucleotide labeling is highly desirable. In this work, we have developed a facile and streamlined source of recombinant enzymes from the pentose phosphate pathway for making such labeled nucleotides. The Escherichia coli (E. coli) genes encoding ribokinase (RK), adenine phosphoribosyltransferase (APRT), xanthine/guanine phosphoribosyltransferase (XGPRT), and uracil phosphoribosyltransferase (UPRT) were sub-cloned into pET15b vectors. All four constructs together with cytidine triphosphate synthetase (CTPS) and human phosphoribosyl pyrophosphate synthetase isoform 1 (PRPPS) were transformed into the E. coli BL21(AI) strain for protein over-expression. The enzyme preparations were purified to >90% homogeneity by a one-step Ni-NTA affinity chromatography, without the need of a further size-exclusion chromatography step. We obtained yields of 1530, 22, 482, 3120, 2120 and 2280 units of activity per liter of culture for RK, PRPPS, APRT, XGPRT, UPRT and CTPS, respectively; the specific activities were found to be 70, 22, 21, 128, 144 and 113 U/mg, respectively. These specific activities of these enzyme constructs are comparable to or higher than those previously reported. In addition, both the growth conditions and purification protocols have been streamlined so that all the recombinant proteins can be expressed, purified and characterized in at most 2 days. The availability and reliability of these constructs should make production of fully and site-specific labeled nucleotides for making labeled RNA accessible and straightforward, to facilitate high-resolution NMR spectroscopic and other biophysical studies.
RNA 比以往任何时候都更被视为未来的生物分子，因其功能的多样性和复杂的三维折叠结构。为了通过核磁共振（NMR）光谱有效地研究它们，结构生物学家需要应对大型 RNA 的光谱拥挤和信号快速衰减这两个关键挑战。稳定同位素核苷酸标记是解决重叠问题的一种有吸引力的方法。因此，非常需要开发有效的核苷酸标记方法。在这项工作中，我们开发了一种简便且简化的来自磷酸戊糖途径的重组酶来源，用于制备此类标记核苷酸。将编码核糖激酶（RK）、腺嘌呤磷酸核糖转移酶（APRT）、黄嘌呤/鸟嘌呤磷酸核糖转移酶（XGPRT）和尿嘧啶磷酸核糖转移酶（UPRT）的大肠杆菌（E. coli）基因亚克隆到 pET15b 载体中。将所有这四种构建体与胞苷三磷酸合成酶（CTPS）和人磷酸核糖焦磷酸合成酶同工型 1（PRPPS）一起转化到大肠杆菌 BL21(AI)菌株中进行蛋白质的过量表达。通过一步 Ni-NTA 亲和色谱将酶制剂纯化至>90%的纯度，无需进一步的尺寸排阻色谱步骤。我们每升培养物分别获得了 1530、22、482、3120、2120 和 2280 单位活性的 RK、PRPPS、APRT、XGPRT、UPRT 和 CTPS；发现其比活性分别为 70、22、21、128、144 和 113 U/mg。这些酶构建体的这些比活性与先前报道的相当或更高。此外，生长条件和纯化方案都已简化，以便所有重组蛋白最多可在 2 天内表达、纯化和表征。这些构建体的可用性和可靠性应使生产用于制备标记 RNA 的完全和位点特异性标记核苷酸变得容易且直接，以促进高分辨率 NMR 光谱和其他生物物理研究。
