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Merck

A novel probe to assess cytosolic entry of exogenous proteins.

Nature communications (2018-08-08)
Qiao Lu, Jeff E Grotzke, Peter Cresswell
摘要

Dendritic cells use a specialized pathway called cross-presentation to activate CD8+ T cells by presenting peptides from exogenous protein antigens on major histocompatibility complex class I molecules. Considerable evidence suggests that internalized antigens cross endocytic membranes to access cytosolic proteasomes for processing. The mechanism of protein dislocation represents a major unsolved problem. Here we describe the development of a sensitive reporter substrate, an N-glycosylated variant of Renilla luciferase fused to the Fc region of human IgG1. The luciferase variant is designed to be enzymatically inactive when glycosylated, but active after the asparagine to aspartic acid conversion that occurs upon deglycosylation by the cytosolic enzyme N-glycanase-1. The generation of cytosolic luminescence depends on internalization, deglycosylation, the cytosolic AAA-ATPase VCP/p97, and the cytosolic chaperone HSP90. By incorporating a T cell epitope into the fusion protein, we demonstrate that antigen dislocation into the cytosol is the rate limiting step in cross-presentation.

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Sigma-Aldrich
Iscove 改良杜氏培养基, liquid, sterile-filtered, With sodium bicarbonate, without L-glutamine, suitable for cell culture, suitable for hybridoma
Sigma-Aldrich
丙酸钠, ≥99.0%
Sigma-Aldrich
链球菌溶血素O 来源于化脓链球菌, lyophilized powder, Protein ~3 % by Lowry, 25,000-50,000 U/vial