果蝇吡咯啉-5-羧酸还原酶的表达、纯化及理化特点

Purification and Characterization of Pyrroline-5-carboxylate Reductase from Drosophila melanogaster

吡咯啉-5-羧酸还原酶(P5CRs)是普遍存在于原核和真核生物中的一种重要管家蛋白,其主要的功能是催化吡咯啉-5-羧酸(P5C)转化为脯氨酸,同时将NAD(P)H氧化为NAD(P)+。为揭示果蝇P5CR的聚合形式、酶学性质及晶体结构,提取了果蝇的总RNA,通过逆转录获得了cDNA,进而通过PCR获得了编码果蝇P5CR的cDNA片段;将此片段连接到pET-28a(+)载体上,在大肠杆菌(Escherichia coli)中得到了高效表达,依次经过硫酸铵分级沉淀、亲和层析及凝胶过滤层析得到了适合晶体生长的高纯度蛋白;通过EGS交联实验和凝胶过滤层析检测了P5CR在溶液中的聚合形式;应用分光光度法测定了果蝇P5CR的酶活性参数;采用悬滴气相扩散法筛选了P5CR的结晶条件并进行了优化。实验结果:(1)纯化后的蛋白样品经SDS-PAGE检测,结果显示,凝胶上已基本观察不到杂蛋白质条带,说明目的蛋白质纯度较高;(2)果蝇P5CR在溶液中的基本存在形式是十聚体,在此基础上可形成更大的聚合形式;(3)果蝇P5CR参与抗癌药物硫代脯氨酸的代谢,在该逆向反应中最适pH为高碱性,该酶在45℃的半衰期约15分钟;(4)筛选得到了果蝇P5CR的初步结晶条件(0.2mol/L Ammonium phosphate dibasic,20%PEG3350 pH 8.0)。

P5CRs exist in every domain of life, including eukarya, bacteria and archea. They catalyze the conversion from Al-pyrroline-5-carboxylate （P5C） to proline with concomitant oxidation of NAD （P）H to NAD （P） ＋ in the final step of proline biosynthesis. In order to provide the theory basis of the function and three- dimensional structure of P5CR, it was performed to research the aggregation form, enzymatic properties and optimal crystallization conditions of P5CR from Drosophila melanogaster. Total RNA from Drosophila melanogaster was isolated, and reverse transcripted into cDNA with random primers. The full-length sequence of the PSCR gene was amplified from the cDNA and comfirmed by sequencing. The coding sequence of P5CR was subcloned into Novagen pET-28a （ ＋ ） vector. The target protein was over-expressed in Escherichia coli and purified to homogeneity by chromatography. The homopolymer was characterized by EGS cross-linking and size exclusion gel filtration chromatography. The thioproline dehydrogenase activity of P5CR was assayed at room temperature using a sensitive spectrophotometric method. The crystallization conditions of P5CR using hanging-drop vapor diffusion method were screened and optimized. The main results were as follows： （1） The method of overexpression and purificaiton of Drosophila melanogaster PSCR was obtained, and the purity of PSCR is high. （2） Drosophila melanogaster PSCR mainly exists in the form of decamer in solution, on this basis, forms homopolymer. （3） PSCR can be involved in the metabolism of thioproline which was considered as a kind of anticancer drug. This enzyme has an optimal alkaline pH in this reversible reaction and is thermo stable with a half-life of approximately 15 min at 45℃. （4） One remarkable crystallization condition was obtained （0.2mol/L Ammonium phosphate dibasic, 20% PEG3350, and pH 8.0）.