A METHOD FOR INCREASING THE YIELD OF PROPERLY FOLDED RECOMBINANT FUSION PROTEINS - SINGLE-CHAIN IMMUNOTOXINS FROM RENATURATION OF BACTERIAL INCLUSION-BODIES
A METHOD FOR INCREASING THE YIELD OF PROPERLY FOLDED RECOMBINANT FUSION PROTEINS - SINGLE-CHAIN IMMUNOTOXINS FROM RENATURATION OF BACTERIAL INCLUSION-BODIES
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DOI：
10.1016/0003-2697(92)90433-8
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发表时间：
1992-09-01
影响因子：
2.9
通讯作者：
BRINKMANN, U
中科院分区：
文献类型：
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作者：
BUCHNER, J;PASTAN, I;BRINKMANN, U
大肠杆菌产生的许多蛋白质积聚在包涵体中。我们系统地评估了影响来自含有重组单链免疫毒素 B3(Fv)-PE38KDEL 的细菌包涵体的酶活性分子的重折叠和复性的参数。该重组分子由单克隆抗体 B3 (B3(Fv)) 的可变结构域与假单胞菌外毒素 A (PE38KDEL) 的截短突变体融合。这种免疫毒素在体外杀死癌细胞，导致动物肿瘤模型中的肿瘤消退，并且正在被开发为抗癌治疗剂（Brinkmann 等人，1991，Proc. Natl. Acad. Sci. USA 88, 8616–8620）。与许多其他重组蛋白一样，B3(Fv)-PE38KDEL 在大肠杆菌中以包涵体形式产生，必须变性并重新折叠才能具有活性。这需要正确的折叠、天然二硫键的形成以及不同结构域的关联。所有这些步骤都强烈依赖于所使用的复性条件。通过添加还原和氧化硫醇试剂以促进二硫键形成以及添加稳定剂（例如L-精氨酸）获得最佳重折叠条件。此外，通过逐步将变性和还原的蛋白质添加到重折叠溶液中，克服了由于其在高浓度下倾向于聚集而在低蛋白质浓度下重新激活蛋白质的必要性。这种方法对于生产其他重组蛋白的活性形式应该有用。
Many proteins produced in Escherichia coli accumulate in inclusion bodies. We have systematically evaluated the parameters that affect the refolding and renaturation of enzymatically active molecules from bacterial inclusion bodies containing a recombinant single-chain immunotoxin, B3(Fv)-PE38KDEL. This recombinant molecule is composed of the variable domains of monoclonal antibody B3 (B3(Fv)) fused to a truncated mutant form of Pseudomonas exotoxin A (PE38KDEL). This immunotoxin kills carcinoma cells in vitro, causes tumor regression in animal tumor models, and is being developed as an anti-cancer therapeutic agent (Brinkmann et al., 1991, Proc. Natl. Acad. Sci. USA 88, 8616–8620). Like many other recombinant proteins, B3(Fv)-PE38KDEL is produced in E. coli in inclusion bodies and must be denatured and refolded to become active. This requires correct folding, formation of native disulfide bonds, and the association of different domains. All these steps are strongly dependent on the renaturation conditions used. Optimum conditions of refolding were obtained by the addition of reduced and oxidized thiol reagents to promote disulfide bond formation and the addition of a labilizing agent such as l-arginine. Furthermore, the necessity to reactivate proteins at low protein concentrations due to its tendency to aggregate at high concentrations was overcome by a step-by-step addition of denatured and reduced protein into the refolding solution. This approach should be useful for the production of active forms of other recombinant proteins.