Nicholls P J, Johnson V G, Andrew S M, Hoogenboom H R, Raus J C, Youle R J
Biochemistry Section, National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 Mar 5;268(7):5302-8.
Chimeric proteins consisting of a fusion between binding-deficient mutants of diphtheria toxin (DT) or Pseudomonas exotoxin A (PE) and a single-chain antibody (E6 sFv) against the human transferrin receptor (TfnR) were expressed in a rabbit reticulocyte lysate system. Molecules utilizing PE40 (the carboxyl terminus 40 kDa of PE, lacking the binding domain) exhibited significant E6 sFv-mediated, cell type-specific cytotoxicity (IC50 1 x 10(-10) M) against a human erythroleukemia-derived cell line, K562. In contrast, a fusion protein between the same sFv and a DT mutant, DTM1 (containing two amino acid substitutions in the binding domain [S(508)F, S(525)F]) was not significantly cytotoxic, despite being enzymatically active. A tripartite protein in the form NH2-DTM1-E6 sFv-PE40-COOH exhibited cytotoxicity comparable to that of the PE40-sFv fusion (IC50 1 x 10(-10) M), suggesting that the deficit in activity of DTM1-sFv is not a function of misfolding of the sFv moiety or of a reduced ability to bind TfnR. In contrast to DTM1-E6 sFv, a fusion protein between a second DT mutant, CRM 107 [S(525)F], and the E6 sFv was specifically cytotoxic (IC50 1 x 10(-9) M), and toxicity could be blocked by addition of excess E6 antibody. The cell-free in vitro expression system we describe is rapid and may be used to express functional toxin-sFv fusion proteins. No protein refolding procedures are required, and the technique may be used to express proteins which, due to restrictions imposed on manipulation of toxin-encoding genes in Escherichia coli, could not be produced by more conventional methods.
由白喉毒素（DT）或绿脓杆菌外毒素A（PE）的结合缺陷型突变体与抗人转铁蛋白受体（TfnR）的单链抗体（E6 sFv）融合而成的嵌合蛋白，在兔网织红细胞裂解物系统中表达。利用PE40（PE的羧基末端40 kDa，缺乏结合结构域）的分子对人红白血病衍生细胞系K562表现出显著的E6 sFv介导的细胞类型特异性细胞毒性（IC50为1×10⁻¹⁰ M）。相比之下，相同sFv与DT突变体DTM1（结合结构域中有两个氨基酸替换[S(508)F，S(525)F]）之间的融合蛋白，尽管具有酶活性，但没有显著的细胞毒性。NH₂-DTM1-E6 sFv-PE40-COOH形式的三方蛋白表现出与PE40-sFv融合蛋白相当的细胞毒性（IC50为1×10⁻¹⁰ M），这表明DTM1-sFv活性的缺陷不是sFv部分错误折叠或结合TfnR能力降低的结果。与DTM1-E6 sFv相反，第二个DT突变体CRM 107 [S(525)F]与E6 sFv之间的融合蛋白具有特异性细胞毒性（IC50为1×10⁻⁹ M），并且加入过量的E6抗体可阻断毒性。我们描述的无细胞体外表达系统速度快，可用于表达功能性毒素-sFv融合蛋白。不需要蛋白质重折叠程序，并且该技术可用于表达由于在大肠杆菌中操纵毒素编码基因受到限制而无法通过更传统方法生产的蛋白质。
