GST- or His-tagged recombinant proteins were produced in E. coli using the GST Gene Fusion System (GE Healthcare) and the Probond™ Purification System (Invitrogen) respectively according to the manufacturers’ instructions and extracts were prepared by urea/DTT denaturation and renaturation as described previously [12 (link)]. Critical to reducing background ubiquitination, the recombinant His-tagged p21 was clarified by centrifugation at 100 000 g for 15 min at 4°C and the protein was stored in aliquots of 20 μl at −80°C, which were thawed no more than twice. Proteins prepared in this manner eluted as a well-defined single peak after gel filtration, albeit they were not purified to homogeneity.
Protocol detail
Recombinant Protein Production and Purification
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Centrifugation
E coli
Gel filtration
Gene system
Probond
Proteins
Recombinant proteins
Ubiquitination
Urea
Corresponding Organization :
Other organizations : Cornell University, Memorial Sloan Kettering Cancer Center
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Variable analysis
independent variables
- Purification method (GST Gene Fusion System vs. Probond Purification System)
- Recombinant protein production in E. coli
- Ubiquitination of recombinant proteins
- Denaturation and renaturation protocol
- Centrifugation parameters (100,000 g for 15 min at 4°C)
- Protein storage conditions (-80°C with no more than two freeze-thaw cycles)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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