Document Type

Article

Publication Date

12-2009

Comments

This article has been peer reviewed. It is the authors' final version prior to publication in Biochemical and Biophysical Research Communications Volume 390, Issue 3, December 2009, Pages 662-666. The published version is available at DOI: 10.1016/j.bbrc.2009.10.024. Copyright © Elsevier Inc.

Abstract

Fluorescent proteins are powerful markers allowing tracking expression, intracellular localization, and translocation of tagged proteins but their effects on the structure and assembly of complex extracellular matrix proteins has not been investigated. Here, we analyzed the utility of fluorescent proteins as markers for procollagen VII, a triple-helical protein critical for the integrity of dermal-epidermal junction. DNA constructs encoding a red fluorescent protein-tagged wild type mini-procollagen VII α chain and green fluorescent protein-tagged α chains harboring selected mutations were genetically engineered. These DNA constructs were co-expressed in HEK-293 cells and the assembly of heterogeneous triple-helical mini-procollagen VII molecules was analyzed. Immunoprecipitation and fluorescence resonance energy transfer assays demonstrated that the presence of different fluorescent protein markers at the C-termini of individual α chains neither altered formation of triple-helical molecules nor affected their secretion to the extracellular space. Our study provides a basis for employing fluorescent proteins as tags for complex structural proteins of extracellular matrix.

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