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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)。
Pyrroline-5-carboxylic acid reductase (P5CRs) is an important housekeeping protein that is ubiquitous in prokaryotes and eukaryotes. Its main function is to catalyze the conversion of pyrroline-5-carboxylic acid (P5C) to proline , While NAD (P) H is oxidized to NAD (P) +. To reveal the polymerization form, enzymatic properties and crystal structure of Drosophila P5CR, the total RNA of Drosophila was extracted, cDNA was obtained by reverse transcription, and then the cDNA fragment encoding P5CR of Drosophila was obtained by PCR. The fragment was ligated to pET -28a (+) vector was successfully expressed in Escherichia coli. The high purity protein suitable for crystal growth was obtained by ammonium sulfate fractionation, affinity chromatography and gel filtration chromatography. The polymerization of P5CR in solution was detected by cross-linking experiments and gel filtration chromatography. The P5CR enzyme activity parameters of Drosophila melanogaster were determined by spectrophotometry. The crystallization conditions of P5CR were screened by hanging drop gas diffusion method and optimized. The results showed that: (1) The purified protein samples were detected by SDS-PAGE. The results showed that the hybrid protein bands were not observed on the gel, indicating that the purity of the target protein was high. (2) The Drosophila P5CR in solution (3) Drosophila P5CR is involved in the metabolism of thio-proline, an anticancer drug, and the optimum pH in this reverse reaction is overbased , The half-life of the enzyme at 45 ℃ for about 15 minutes; (4) screening of the initial crystallization conditions of P5CR Drosophila (0.2mol / L Ammonium phosphate dibasic, 20% PEG3350 pH 8.0).