Hartman J, Daram P, Frizzell R A, Rado T, Benos D J, Sorscher E J
Departments of Medicine, Division of Hematology and Oncology, UAB Station, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
Biotechnol Bioeng. 1992 Apr 5;39(8):828-32. doi: 10.1002/bit.260390805.
Prokaryotic expression of polypeptides as fusion proteins with glutathione-S-transferase has recently been reported as a one-step means of purifying recombinant protein. The usefulness of the glutathione-S-transferase/glutathione-agarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insoluble form by Escherichia coli. We have found that for 5 separate fusion proteins containing glutathione-S-transferase and different domains of the large cystic fibrosis transmembrane conductance regulator, all were packaged in insoluble form by E. coli. Insolubility of these products made them inaccessible to one-step purification utilizing this scheme requires proper folding of recombinant glutathione-S-transferase to allow recognition on glutathione affinity agarose, we investigated the suitability of several alternative approaches for converting insoluble recombinant fusion proteins to a soluble form amenable to glutathione-agarose affinity purification. Low-temperature induction of fusion protein synthesis, but not incubation with anion-exchange resins, led to improved one-step purification of glutathione-S-transferase fusion proteins from E. coli cell lysate using mild, nondenaturing conditions. Solubilization in 8 mol/L urea, but not with other chaotropic agents or detergents, also allowed preparative yields of affinity-purified fusion protein. These techniques increase the usefulness of this recombinant protein purification scheme, and should be broadly applicable to diverse polypeptides synthesized as fusions with glutathione-S-transferase.
最近有报道称，将多肽作为与谷胱甘肽 - S - 转移酶的融合蛋白进行原核表达是纯化重组蛋白的一种一步法。然而，谷胱甘肽 - S - 转移酶/谷胱甘肽 - 琼脂糖系统的实用性受到大肠杆菌频繁以不溶性形式合成重组蛋白的显著限制。我们发现，对于5种分别含有谷胱甘肽 - S - 转移酶和大囊性纤维化跨膜传导调节因子不同结构域的融合蛋白，所有这些蛋白都被大肠杆菌以不溶性形式包裹。这些产物的不溶性使得它们无法通过该方案进行一步纯化，因为该方案需要重组谷胱甘肽 - S - 转移酶正确折叠以允许在谷胱甘肽亲和琼脂糖上被识别，我们研究了几种将不溶性重组融合蛋白转化为适合谷胱甘肽 - 琼脂糖亲和纯化的可溶性形式的替代方法的适用性。低温诱导融合蛋白合成，而不是与阴离子交换树脂孵育，可在温和的非变性条件下从大肠杆菌细胞裂解物中提高谷胱甘肽 - S - 转移酶融合蛋白的一步纯化效果。在8 mol/L尿素中溶解，而不是用其他变性剂或去污剂，也能获得亲和纯化融合蛋白的制备产量。这些技术提高了这种重组蛋白纯化方案的实用性，并且应该广泛适用于作为与谷胱甘肽 - S - 转移酶融合体合成的各种多肽。
