de Marco A, Volrath S, Bruyere T, Law M, Fonné-Pfister R
LD Biochemistry Unit, Novartis Crop Protection AG, Basel, CH-4002, Switzerland.
Protein Expr Purif. 2000 Oct;20(1):81-6. doi: 10.1006/prep.2000.1274.
The clone corresponding to maize plastidic protoporphyrinogen IX oxidase (PPO) has been isolated by functional complementation and inserted into a pET16b vector for expression in Escherichia coli. Recombinant PPO was purified by standard affinity chromatography using a metal chelating resin. Two contaminants copurified with recombinant PPO and were identified as GroEL and DnaK. Since chaperone binding to hydrophobic regions of the protein is regulated by ATP availability, an ATP washing step was introduced prior to elution of the recombinant protein from an affinity column. This washing step selectively removed both chaperones and allowed the recovery of pure PPO. Coexpression of PPO and GroELS resulted in a sixfold increase of soluble PPO yield, suggesting that bacterial chaperones could be limiting during the folding of the heterologous protein. However, a portion of PPO was still found in the insoluble fraction. Buffer containing the GroEL and DnaK enabled resuspension of PPO from the insoluble fraction but failed to enhance refolding of the denaturated protein. Attempts to increase the amount of soluble PPO using a thioredoxin-PPO fusion protein were not successful. Initial characterization of the recombinant PPO found that it possessed a high V(max), an elevated affinity for substrate, and an elevated sensitivity to PPO inhibitor herbicides compared to previous reports.
通过功能互补分离出了与玉米质体原卟啉原IX氧化酶（PPO）对应的克隆，并将其插入pET16b载体中以在大肠杆菌中表达。使用金属螯合树脂通过标准亲和层析纯化重组PPO。两种污染物与重组PPO共纯化，并被鉴定为GroEL和DnaK。由于伴侣蛋白与蛋白质疏水区域的结合受ATP可用性的调节，因此在从亲和柱洗脱重组蛋白之前引入了ATP洗涤步骤。该洗涤步骤选择性地去除了两种伴侣蛋白，并使得能够回收纯PPO。PPO与GroELS的共表达使可溶性PPO产量提高了六倍，这表明细菌伴侣蛋白在异源蛋白折叠过程中可能是限制性因素。然而，仍有一部分PPO存在于不溶性部分中。含有GroEL和DnaK的缓冲液能够使不溶性部分中的PPO重新悬浮，但未能增强变性蛋白的复性。使用硫氧还蛋白-PPO融合蛋白增加可溶性PPO量的尝试未成功。重组PPO的初步表征发现，与先前的报道相比，它具有较高的V(max)、对底物的亲和力升高以及对PPO抑制剂除草剂的敏感性升高。
