Burks E A, Iverson B L
Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
Biotechnol Prog. 1995 Jan-Feb;11(1):112-4. doi: 10.1021/bp00031a017.
We have isolated milligram quantities of active single chain antibody from the insoluble fraction of Escherichia coli cultures. The system relies on high-level expression from a T7 RNA polymerase-directed gene construct, 8 M urea to dissolve the desired protein out of the insoluble fraction, presumably inclusion bodies, isolation and concentration of the desired protein by nickel chelate [IDA-Ni(II)] immobilized metal-ion affinity chromatography (IMAC), and removal of urea from column fractions by dialysis directly into storage buffer. Routinely, about 50% of the protein loaded onto an IMAC column is recovered as single chain Fv at a concentration of approximately 0.7 mg/mL. As little as 3 days are required to obtain 10 mg of final product when starting with an overnight inoculum.
我们已从大肠杆菌培养物的不溶性部分中分离出毫克量的活性单链抗体。该系统依赖于T7 RNA聚合酶指导的基因构建体的高水平表达，用8M尿素从可能是包涵体的不溶性部分中溶解所需蛋白质，通过镍螯合[IDA-Ni(II)]固定金属离子亲和色谱法（IMAC）分离和浓缩所需蛋白质，以及通过直接透析到储存缓冲液中从柱级分中去除尿素。通常，加载到IMAC柱上的蛋白质约50%以浓度约为0.7mg/mL的单链Fv形式回收。从过夜接种物开始，只需3天就能获得10mg的最终产物。
