Inhibition of cytoplasmic antigen, glucose-6-phosphate dehydrogenase, by V-H-C(H)1, an intracellular Fd fragment antibody derived from a semisynthetic Fd fragment phage display library

A library of Fd fragment antibody binding proteins was created by random mu tation of 15 nucleotides within the CDRIII region of the immunoglobulin hea vy chain gene and displayed as Fd coat protein fusion constructs of M13 pha ge. The library was screened for those V-H binding sites that bound glucose -6-phosphate dehydrogenase (G6PD). One isolate (DH27(bp)) inhibited G6PD ac tivity by 85%. The DH27(bp) gene was reengineered, placed in a eukaryotic e xpression 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 reverte d the cell to full G6PD activity: The intracellular dynamics of the G6PD/DH 27(bp) complex showed that when the proteasomes of cells expressing DH27(bp ) were inhibited (N-acetyl-Leu-Leu-norleucinal or lactacystin) G6PD activit y increased. Metabolic labelling of newly synthesized IPTG-induced proteins during/absence of proteasomal inhibitors showed that both G6PD and DH27(bp ) are signaled for degradation when the intracellular complex is formed, fu rthermore, semi-quantitative RT/PCR demonstrated that GGPD mRNA is upregula ted over the time course of G6PD inactivation by DH27(bp) Fd binding protei n. 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 t he Fd expression is repressed the activity of intracellular targeted protei n can revert to normal. (C) 1999 Academic Press.