Generating a Glutathione S-Transferase (GST) Fusion Protein

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Producing A GST Fusion Protein

1. Make sure that your gene does not contain any of the restriction sites that you choose to cut out your gene with.
2. Perform PCR of the gene that you wish to fuse with GST and of GST, removing the stop codon from the gene if inserting GST to the 3' end of the gene. Remember to generate restriction sites on either site of each gene so that you can ligate it into a suitable vector.
3. Digest out the restriction sites from a vector, and purify out the vector to remove the DNA between the restriction sites. Also digest the introduced restriction sites at the 5' and 3' ends of the PCR products.
4. Perform a ligation with the genes produced by PCR and the target vector.
5. Transform the desired amount of ligated product into competent bacterial cells.
6. Select the desired colonies and grow up the bacteria in overnight cultures.
7. Purify the plasmid (eg can use commercial kits).
8. Store the purified plasmid at -80 degrees C or -20 degrees C in aliquots.
9. Express the subcloned fusion gene as a protein either in bacteria such as E. coli via induction, or in higher-level organisms such as mammals, where you can perform transfection.
10. If desired, purify the expressed GST fusion protein.