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Updated: Jul 5, 2026

Studying Protein Import into Chloroplasts Using Protoplasts
Published on: December 10, 2018
Development and optimization of an in vitro chloroplastic protein import assay using recombinant proteins
Hitoshi Inoue1, R M Udayangani Ratnayake, Hiroshi Nonami
1The United Graduate School of Agricultural Science, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan.
Abstract:
The in vitro protein import experiment is one of the most important techniques for determining protein localization. For chloroplastic proteins, proteins of interest are incubated with isolated chloroplasts in the presence of energy sources. Radio-labeled proteins synthesized either in vitro or in vivo have been widely used as substrate proteins. Here we report our development of the protein import assay system in which non-radio-labeled proteins, overexpressed in Escherichia coli, were applied. In this system, substrate proteins were designed to carry epitope-tags, thus allowing analysis of imported proteins by SDS-PAGE, followed by immunoblotting to detect these tags. Furthermore, the imported proteins were found to be incorporated into their native form. These observations indicated that recombinant proteins were imported into chloroplasts and folded correctly. Therefore, this assay system could represent another valuable tool for determining protein localization.
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