Kenten J, Helm B, Ishizaka T, Cattini P, Gould H
Proc Natl Acad Sci U S A. 1984 May;81(10):2955-9. doi: 10.1073/pnas.81.10.2955.
A fragment of the cloned gene for the human myeloma ND epsilon chain, coding for the second, third, and fourth domains of the immunoglobulin, has been coupled to the tryptophan control region of an expression plasmid and subcloned in Escherichia coli. Induction of gene expression results in the synthesis of the expected, antigenically active polypeptide of Mr 40,000, which constitutes 18% of total bacterial protein and yields 55 mg/liter of culture. The immunoglobulin, which is aggregated and packed into large inclusion bodies within the bacterial cell, can be dissolved by denaturing solvents and purified by affinity chromatography using anti-IgE Sepharose. Reduced monomeric chains assemble spontaneously into dimers. On assay to measure the inhibition of binding of 125I-labeled human E myeloma protein to Fc epsilon receptors on cultured human basophils, the cloned gene product exhibited 20% of the activity of the native protein.
编码免疫球蛋白第二、第三和第四结构域的人骨髓瘤NDε链克隆基因片段，已与表达质粒的色氨酸控制区连接，并亚克隆到大肠杆菌中。基因表达的诱导导致合成预期的、抗原活性的40000 Mr多肽，其占细菌总蛋白的18%，每升培养物产量为55 mg。免疫球蛋白在细菌细胞内聚集并包装成大的包涵体，可通过变性溶剂溶解，并使用抗IgE琼脂糖通过亲和层析纯化。还原的单体链自发组装成二聚体。在测定125I标记的人E骨髓瘤蛋白与培养的人嗜碱性粒细胞上的Fcε受体结合的抑制作用时，克隆基因产物表现出天然蛋白20%的活性。
