The recombinant GST- and Hisx6-fused proteins were expressed in E. coli (BL21, DE3) as described previously.10 (link), 42 (link), 53 (link), 54 (link) The resulting recombinant proteins were purified using Sepharose 4 Fast Flow purification resin (GE Healthcare Life Sciences) for GST tagged and the TALON CellThru Resin (ClonTech) for the Hisx6-peptide fused proteins according to the instructions of the manufacturers. The GST was removed from the target proteins by thrombin (GE Healthcare Life Sciences) cleavage, while the GST-fusion proteins still bound to the purification resin.
Protocol detail
Purification of Recombinant Fusion Proteins
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Cleavage
E coli
Peptide
Proteins
Proteins target
Recombinant proteins
Resin
Sepharose
Talon
Thrombin
Corresponding Organization : University of Cincinnati Medical Center
Other organizations : Purdue University West Lafayette, University of Alberta
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Variable analysis
independent variables
- Expression of recombinant GST- and Hisx6-fused proteins in E. coli (BL21, DE3)
- Purification of the recombinant proteins using Sepharose 4 Fast Flow purification resin (for GST tagged) and TALON CellThru Resin (for Hisx6-peptide fused proteins)
- Removal of GST from the target proteins by thrombin (GE Healthcare Life Sciences) cleavage
- Manufacturer's instructions for protein purification
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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