KRT8 Human, GST

Cytokeratin 8 is a member of the type II keratin family, which is clustered on the long arm of chromosome 12. These keratins are essential components of the cytoskeleton in epithelial cells, providing structural support and contributing to various cellular functions, including cell signaling, apoptosis, and stress response. CK8 is particularly significant in non-squamous epithelia and is present in the majority of adenocarcinomas and ductal carcinomas .

The recombinant form of Cytokeratin 8, tagged with Glutathione S-transferase (GST), is produced using various expression systems, including E. coli and wheat germ. The GST tag facilitates the purification process through affinity chromatography, ensuring high purity and yield of the recombinant protein.

1. Expression in E. coli: The gene encoding CK8 is cloned into an expression vector containing the GST tag. The recombinant plasmid is then transformed into E. coli cells, which are cultured under optimal conditions to induce protein expression. The cells are harvested, lysed, and the recombinant CK8-GST fusion protein is purified using glutathione affinity chromatography .

2. Expression in Wheat Germ: An alternative method involves the in vitro wheat germ expression system. This system preserves the correct conformational folding of the protein, which is crucial for its biological function. The recombinant CK8-GST protein is purified similarly using glutathione affinity chromatography .

Cytokeratin 8 undergoes various post-translational modifications, including phosphorylation, glycosylation, and ubiquitination, which regulate its function and interactions with other proteins. These modifications can influence the stability, solubility, and assembly of CK8 filaments.

1. Phosphorylation: CK8 is phosphorylated at multiple serine residues, which modulates its interaction with CK18 and other binding partners. Phosphorylation plays a critical role in the dynamic reorganization of the cytoskeleton during cell division, migration, and apoptosis.

2. Glycosylation: Glycosylation of CK8 can affect its solubility and stability. This modification is essential for the proper folding and function of the protein within the cellular environment.

3. Ubiquitination: Ubiquitination targets CK8 for degradation via the proteasome pathway. This process is crucial for maintaining the balance of cytokeratin levels within the cell and preventing the accumulation of damaged or misfolded proteins.

Recombinant CK8 tagged with GST is widely used in research to study the structure and function of intermediate filaments, as well as their role in various diseases, including cancer. The GST tag allows for easy purification and detection of the protein in various assays, such as Western blotting, ELISA, and immunoprecipitation .