Kipriyanov S M, Moldenhauer G, Little M
Diagnostics and Experimental Therapy Programme, German Cancer Research Center, Heidelberg.
J Immunol Methods. 1997 Jan 15;200(1-2):69-77. doi: 10.1016/s0022-1759(96)00188-3.
We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter. The scFv was directed into the periplasmic space by a pelB leader sequence. Addition of sucrose to the medium gave a 15-25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80-150-fold (16.5 mg/l) for shake-flask cultures. Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium. We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer. The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter. Lowering the incubation temperature below 20 degrees C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium. An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3. The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry. This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.
我们研究了生长和诱导条件对在野生型乳糖启动子控制下的大肠杆菌中可溶性单链Fv抗体片段产生的影响。单链Fv通过pelB前导序列被导向周质空间。向培养基中添加蔗糖，对于细菌摇管培养，可溶性单链Fv-phOx的产量提高了15 - 25倍（3.0毫克/升），对于摇瓶培养提高了80 - 150倍（16.5毫克/升）。在0.4 M蔗糖存在下进行摇瓶培养时，大量单链Fv释放到培养基中。我们发现，通过简单改变培养条件和诱导剂浓度，单链Fv可以在周质中积累或分泌到培养基中。可溶性抗体片段与不溶性单链Fv聚集体之间的比例被证明取决于启动子的强度。将培养温度降低到20摄氏度以下对周质中可溶性抗体片段的产量没有影响，但它们不再分泌到培养基中。描述了针对T细胞表面抗原CD3的可溶性单链Fv在摇瓶培养中的高水平生产以及通过固定化金属亲和色谱（IMAC）进行一步纯化的实例。通过流式细胞术证明了纯化的抗CD3单链Fv的生物活性。该方法对于在小规模培养中对大量克隆进行功能筛选应该特别有用。
