Removal of endotoxin from recombinant protein preparations
Removal of endotoxin from recombinant protein preparations
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DOI：
10.1016/s0009-9120(97)00049-0
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发表时间：
1997-08-01
影响因子：
2.8
通讯作者：
Jackowski, E
中科院分区：
文献类型：
--
作者：
Liu, SG;Tobias, R;Jackowski, E
目的：建立一种有效的去除大规模大肠杆菌内毒素的方法。设计和方法：采用Triton X-114相分离、利用固定化多粘菌素B或固定化组氨酸的亲和层析，从重组CK-BB、CK-MB、CK-MM、肌红蛋白和心肌肌钙蛋白I的纯化制剂中去除内毒素。通过鲎变形细胞溶解物凝胶法测定内毒素水平。通过BIAcore（TM）分析，使用一组内部产生的单克隆抗体，并通过Stratus（R）Flu orometric Analyzer测定这些蛋白质制剂的免疫活性。在肌钙蛋白I的情况下，BIAcore（TM）也被用来测量肌钙蛋白C interactions.Results：相分离与Triton X-114是最有效的方法，在减少蛋白制剂中存在的内毒素的量相比，无论是多粘菌素B或组氨酸亲和层析。使用Triton X-114，内毒素水平降低超过99%，去除内毒素后蛋白质回收率超过90%。当用一组单克隆抗体检测时，所有三种去除内毒素的方法对大多数蛋白质的免疫活性没有有害影响。肌钙蛋白I也保留了其在Ca 2+存在下与肌钙蛋白C结合的能力。重组CK-BB和CK-MM在E.结论：TritonX-114相分离法是去除重组蛋白中内毒素的最有效方法。
Objectives: To develop an effective method to remove endotoxin from large scale E. coli recombinant protein purifications.Design and Methods: Triton X-114 phase separation, affinity chromatography utilizing immobilized polymyxin B or immobilized histidine, were used to remove endotoxin from purified preparations of recombinant CK-BB, CK-MB, CK-MM, myoglobin, and cardiac troponin I. Endotoxin levels were measured by a Limulus Amebocyte Lysate gel-clot assay. The immunoactivity of these protein preparations was determined by BIAcore(TM) analysis using a panel of in-house generated monoclonal antibodies and by a Stratus(R) Flu orometric Analyzer. In the case of troponin I, the BIAcore(TM) was also utilized to measure troponin C interactions.Results: Phase separation with Triton X-114 was the most effective method in reducing the amount of endotoxin present in the protein preparations compared to either polymyxin B or histidine affinity chromatography. With Triton X-114, the reduction in endotoxin levels was greater than 99% and recovery of the proteins after endotoxin removal was greater than 90%. All three procedures for removing endotoxin had no deleterious effects on the immunoactivity of majority proteins when tested with a panel of monoclonal antibodies. Troponin I also retained its ability to bind to troponin C in the presence of Ca2+. Recombinant CK-BB and CK-MM which were expressed in the soluble fraction of E. coli cell lysates, contained significantly higher endotoxin levels than recombinant CK-MB, myoglobin and cardiac troponin I which were expressed in the form of inclusion bodies.Conclusion: Of the three methods tested, Triton X-114 phase separation was the most effective way of removing endotoxin from recombinant proteins.