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首页> 外文期刊>Journal of Molecular Biology >Inhibition of cytoplasmic antigen, glucose- 6-phosphate dehydrogenase, by VH-CH1, an intracellular Fd fragment antibody derived from a semisynthetic Fd fragment phage display library.
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Inhibition of cytoplasmic antigen, glucose- 6-phosphate dehydrogenase, by VH-CH1, an intracellular Fd fragment antibody derived from a semisynthetic Fd fragment phage display library.

机译：VH-CH1对细胞质抗原，葡萄糖-6-磷酸脱氢酶的抑制，VH-CH1是一种来自半合成Fd片段噬菌体展示文库的细胞内Fd片段抗体。

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摘要

A library of Fd fragment antibody binding proteins was created by random mutation of 15 nucleotides within the CDRIII region of the immunoglobulin heavy chain gene and displayed as Fd coat protein fusion constructs of M13 phage. The library was screened for those VHbinding sites that bound glucose-6-phosphate dehydrogenase (G6PD). One isolate (DH27bp) inhibited G6PD activity by 85 %. The DH27bpgene was re-engineered, placed in a eukaryotic expression vector having an isopropyl-beta-delta-thiogalactopyranoside (IPTG) inducible promoter, and transfected and then expressed in Chinese hamster V79 cells. G6PD activity was completely inhibited. Removal of IPTG reverted the cell to full G6PD activity. The intracellular dynamics of the G6PD/DH27bpcomplex showed that when the proteasomes of cells expressing DH27bpwere inhibited (N -acetyl-Leu-Leu-norleucinal or lactacystin) G6PD activity increased. Metabolic labelling of newly synthesized IPTG-induced proteins during/absence of proteasomal inhibitors showed that both G6PD and DH27bpare signaled for degradation when the intracellular complex is formed. Furthermore, semi-quantitative RT/PCR demonstrated that G6PD mRNA is upregulated over the time course of G6PD inactivation by DH27bpFd binding protein. These effects were not observed in those cells expressing a non-mutated Fd (UMHC) or in IPTG-treated non-transduced V79 cells. Our results demonstrate that an Fd-based intracellular binding protein can find and disable the function of a specific intracellular target and once the Fd expression is repressed the activity of intracellular targeted protein can revert to normal. Copyright 1999 Academic Press.
机译：通过随机突变免疫球蛋白重链基因CDRIII区域内的15个核苷酸，创建了Fd片段抗体结合蛋白文库，并显示为M13噬菌体的Fd外壳蛋白融合构建体。筛选该文库中结合葡萄糖-6-磷酸脱氢酶（G6PD）的那些VH结合位点。一种分离株（DH27bp）抑制G6PD活性达85％。重新设计DH27bp基因，将其置于具有异丙基-β-δ-硫代半乳糖吡喃糖苷（IPTG）诱导型启动子的真核表达载体中，并转染后在中国仓鼠V79细胞中表达。 G6PD活性被完全抑制。 IPTG的去除使细胞恢复到完整的G6PD活性。 G6PD / DH27bp复合体的细胞内动力学表明，当表达DH27bp的细胞的蛋白酶体被抑制时（N-乙酰-Leu-Leu-神经氨酸或乳腺素），G6PD活性增加。蛋白酶体抑制剂存在/缺乏期间新合成的IPTG诱导蛋白的代谢标记显示，当细胞内复合物形成时，G6PD和DH27bp均发出降解信号。此外，半定量RT / PCR证实，DH6bpFd结合蛋白会在G6PD失活的时间过程中上调G6PD mRNA。在表达非突变Fd（UMHC）的细胞或IPTG处理的非转导V79细胞中未观察到这些作用。我们的结果表明，基于Fd的细胞内结合蛋白可以发现并破坏特定细胞内靶标的功能，一旦Fd表达被抑制，细胞内靶蛋白的活性便可以恢复正常。版权所有1999，学术出版社。

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