Batra J K, Kasturi S, Gallo M G, Voorman R L, Maio S M, Chaudhary V K, Pastan I
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Mol Immunol. 1993 Mar;30(4):379-86. doi: 10.1016/0161-5890(93)90067-l.
CD4-PE40 is a recombinant toxin containing the binding domain of CD4 and a mutant form of Pseudomonas exotoxin A from which the cell binding domain has been removed. To increase the serum half-life of CD4-PE40, we have inserted various portions of the constant domain of human IgG1 into CD4-PE40. The constructs made include CD4-CH2-PE40, CD4-CH3-PE40, CD4-CH1-CH2-PE40 and CD4-CH2-CH3-PE40. The fusion proteins were expressed and purified from E. coli. Insertion of various domains from the constant region of IgG1 did not alter the cytotoxic activity of CD4-PE40; all these molecules were equally cytotoxic to cells expressing gp120 on their surface. However, there was a marked increase in the serum mean residence time of CD4-CH2-PE40 which was 115 min as compared to 47 min for CD4-PE40. Insertion of other domains also increased the half-life of CD4-PE40, however, CD4-CH2-PE40 was found to have the longest mean residence time in the circulation. One possible explanation for the increase in plasma half-life is diminished susceptibility of proteins to proteolysis. It was found that CD4-CH2-PE40 was much more resistant to proteolysis by trypsin than CD4-PE40. We proposed that insertion of the CH2 domain into CD4-PE40 covers up the protease sensitive sites in the molecule, thereby making the molecule less susceptible to degradation. The increase in size and reduced sensitivity to proteases could both be responsible for the increased plasma half-life of CD4-CH2-PE40.
CD4-PE40是一种重组毒素，它包含CD4的结合结构域和一种已去除细胞结合结构域的铜绿假单胞菌外毒素A的突变形式。为了延长CD4-PE40在血清中的半衰期，我们将人IgG1恒定结构域的各个部分插入到CD4-PE40中。构建的产物包括CD4-CH2-PE40、CD4-CH3-PE40、CD4-CH1-CH2-PE40和CD4-CH2-CH3-PE40。这些融合蛋白在大肠杆菌中表达并纯化。从IgG1恒定区插入不同结构域并未改变CD4-PE40的细胞毒性活性；所有这些分子对表面表达gp120的细胞具有同等的细胞毒性。然而，CD4-CH2-PE40在血清中的平均驻留时间显著增加，为115分钟，而CD4-PE40为47分钟。插入其他结构域也延长了CD4-PE40的半衰期，然而，发现CD4-CH2-PE40在循环中的平均驻留时间最长。血浆半衰期增加的一个可能解释是蛋白质对蛋白水解的敏感性降低。发现CD4-CH2-PE40比CD4-PE40对胰蛋白酶的蛋白水解更具抗性。我们推测将CH2结构域插入CD4-PE40中掩盖了分子中的蛋白酶敏感位点，从而使该分子更不易被降解。分子大小的增加和对蛋白酶敏感性的降低都可能是CD4-CH2-PE40血浆半衰期延长的原因。
