Chao B H, Costopoulos D S, Curiel T, Bertonis J M, Chisholm P, Williams C, Schooley R T, Rosa J J, Fisher R A, Maraganore J M
Biogen, Inc., Cambridge, Massachusetts 02142.
J Biol Chem. 1989 Apr 5;264(10):5812-7.
A gene encoding a 113-amino acid, NH2-terminal fragment of CD4, rsT4.113, was constructed and expressed in Escherichia coli under the control of the tryptophan operon promoter. Following induction, rsT4.113 is produced at 5-10% of total E. coli protein, and it is found in inclusion bodies. The protein is purified in two steps under denaturing and reducing conditions. Solubilized rsT4.113 is first purified on a column of Q-Sepharose to remove low molecular weight contaminants and then purified to greater than 95% homogeneity by gel filtration. Renaturation of rsT4.113 is achieved at approximately 20% yield by dilution and dialysis. High performance liquid chromatography analysis of renatured rsT4.113 reveals a less than 15% contaminant of reduced protein. Purified and renatured rsT4.113 contains epitopes for both OKT4a and Leu3a, anti-CD4 monoclonal antibodies which block CD4-gp 120 association, but lacks measurable affinity toward a nonblocking anti-CD4 monoclonal antibody, OKT4. By comparison to a longer form (375 amino acids) of recombinant soluble T4 produced in mammalian cells that contains the entire extracellular domain, rsT4.113 has a comparable affinity for binding to OKT4a and Leu3a in a radioimmunoassay. Analysis of antiviral activity of rsT4.113 demonstrates that the E. coli-derived protein inhibits human immunodeficiency virus-induced syncytium formation with an IC50 of 5-10 micrograms/ml. These data demonstrate that the human immunodeficiency virus-binding domain of CD4 is localized within the NH2-terminal 113 amino acids of CD4 and is contained within a structure homologous to the kappa variable-like domain of immunoglobulins.
构建了一个编码CD4的113个氨基酸的NH2末端片段（rsT4.113）的基因，并在色氨酸操纵子启动子的控制下在大肠杆菌中表达。诱导后，rsT4.113的产量占大肠杆菌总蛋白的5%-10%，且存在于包涵体中。该蛋白在变性和还原条件下分两步纯化。首先将溶解的rsT4.113在Q-Sepharose柱上纯化以去除低分子量污染物，然后通过凝胶过滤纯化至纯度大于95%。通过稀释和透析，rsT4.113的复性产率约为20%。对复性后的rsT4.113进行高效液相色谱分析，结果显示还原蛋白的污染物含量低于15%。纯化和复性后的rsT4.113含有OKT4a和Leu3a这两种抗CD4单克隆抗体的表位，它们可阻断CD4与gp120的结合，但对非阻断性抗CD4单克隆抗体OKT4缺乏可测量的亲和力。与在哺乳动物细胞中产生的包含整个细胞外结构域的较长形式（375个氨基酸）的重组可溶性T4相比，rsT4.113在放射免疫分析中对OKT4a和Leu3a的结合亲和力相当。对rsT4.113的抗病毒活性分析表明，这种大肠杆菌来源的蛋白可抑制人免疫缺陷病毒诱导的合胞体形成，IC50为5-10微克/毫升。这些数据表明，CD4的人免疫缺陷病毒结合结构域位于CD4的NH2末端113个氨基酸内，且包含在与免疫球蛋白κ可变样结构域同源的结构中。
