Zakharova G S, Poloznikov A A, Chubar T A, Gazaryan I G, Tishkov V I
A.N. Bach Institute of Biochemistry, RAS, 119071 Moscow, Russia; Innovations and High Technologies MSU Ltd, 109559 Moscow, Russia.
Innovations and High Technologies MSU Ltd, 109559 Moscow, Russia; M.V. Lomonosov Moscow State University, Chemistry Faculty, Department of Chemical Enzymology, 119899 Moscow, Russia.
Protein Expr Purif. 2015 Sep;113:85-93. doi: 10.1016/j.pep.2015.05.007. Epub 2015 May 15.
Anionic tobacco peroxidase (TOP) is extremely active in chemiluminescence reaction of luminol oxidation without addition of enhancers and more stable than horseradish peroxidase under antibody conjugation conditions. In addition, recombinant TOP (rTOP) produced in Escherichia coli is known to be a perfect direct electron transfer catalyst on electrodes of various origin. These features make the task of development of a high-yield reactivation protocol for rTOP practically important. Previous attempts to reactivate the enzyme from E. coli inclusion bodies were successful, but the reported reactivation yield was only 14%. In this work, we thoroughly screened the refolding conditions for dilution protocol and compared it with gel-filtration chromatography. The impressive reactivation yield in the dilution protocol (85%) was achieved for 8 μg/mL solubilized rTOP protein and the refolding medium containing 0.3 mM oxidized glutathione, 0.05 mM dithiothreitol, 5 mM CaCl2, 5% glycerol in 50 mM Tris-HCl buffer, pH 9.6, with 1 μM hemin added at the 24th hour of incubation. A practically important discovery was a 30-40% increase in the reactivation yield upon delayed addition of hemin. The reactivation yield achieved is one of the highest reported in the literature on protein refolding by dilution. The final yield of purified active non-glycosylated rTOP was ca. 60 mg per L of E. coli culture, close to the yield reported before for tomato and tobacco plants overexpressing glycosylated TOP (60 mg/kg biomass) and much higher than for the previously reported refolding protocol (2.6 mg per L of E. coli culture).
阴离子烟草过氧化物酶（TOP）在鲁米诺氧化的化学发光反应中极其活跃，无需添加增强剂，并且在抗体偶联条件下比辣根过氧化物酶更稳定。此外，已知在大肠杆菌中产生的重组TOP（rTOP）是各种来源电极上完美的直接电子转移催化剂。这些特性使得开发一种高产率的rTOP再活化方案变得至关重要。之前从大肠杆菌包涵体中再活化该酶的尝试取得了成功，但报道的再活化产率仅为14%。在这项工作中，我们全面筛选了稀释方案的复性条件，并将其与凝胶过滤色谱法进行了比较。对于8μg/mL溶解的rTOP蛋白和含有0.3mM氧化型谷胱甘肽、0.05mM二硫苏糖醇、5mM氯化钙、5%甘油的50mM Tris-HCl缓冲液（pH 9.6）的复性培养基，在孵育的第24小时添加1μM血红素，在稀释方案中实现了令人印象深刻的再活化产率（85%）。一个实际重要的发现是延迟添加血红素后再活化产率提高了30 - 40%。所实现的再活化产率是文献中报道的通过稀释进行蛋白质复性的最高产率之一。纯化的活性非糖基化rTOP的最终产量约为每升大肠杆菌培养物60mg，接近之前报道的过表达糖基化TOP的番茄和烟草植株的产量（60mg/kg生物量），并且远高于之前报道的复性方案（每升大肠杆菌培养物2.6mg）。
