Cloning and Expression of GST-Tagged Drosophila Gid8 Protein

Vanessa He

doi:10.58445/rars.4255

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Vanessa He Princeton High School

DOI:

https://doi.org/10.58445/rars.4255

Keywords:

Gid8, GST fusion protein, pGEX4T1, recombinant protein expression, SDS-PAGE, Coomassie staining, Western blot

Abstract

The cloning and expression of specific proteins are classical approaches for biochemical characterization of protein function and regulation. This project aims to clone, express, and purify Gid8 protein, a conserved subunit of the GID (Glucose-Induced Degradation-deficient) complex, in Drosophila Melanogaster. Although Gid8 is highly conserved in eukaryotes, its molecular functions remain poorly understood. The Gid8 coding sequence was amplified by Polymerase Chain Reaction (PCR) and inserted into the pGEX4T1 plasmid vector to generate a construct encoding Glutathione S-Transferase (GST)-Gid8 fusion proteins. The recombinant plasmid was subsequently transformed into E. coli Rosetta cells, and protein expression was induced by the addition of isopropyl beta-D-1-thiogalactopyranoside (IPTG). The GST-tagged Gid8 protein was then isolated using glutathione agarose affinity resin and analyzed by SDS-PAGE with Coomassie staining and Western blotting. These experiments demonstrated the successful cloning, expression, affinity purification, and detection of GST-Gid8, providing a foundation for future biochemical analyses of Gid8 and the GID complex in Drosophila.

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https://www.genscript.com/gsfiles/vector%2dmap/bacteria/pGEX%2d4T%2d1%2epdf?=20231215?1768664770