PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of bmcbiotBioMed Centralsearchsubmit a manuscriptregisterthis articleBMC Biotechnology
 
BMC Biotechnol. 2012; 12: 63.
Published online Sep 18, 2012. doi:  10.1186/1472-6750-12-63
PMCID: PMC3463477
In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins
Jason S Buhrman,1 Jamie E Rayahin,1 Melanie Köllmer,1 and Richard A Gemeinhartcorresponding author1,2,3
1Department of Biopharmaceutical Sciences, University of Illinois, Chicago, IL, 60612-7231, USA
2Department of Bioengineering, University of Illinois, Chicago, IL, 60607-7052, USA
3Department of Ophthalmology and Visual Science, University of Illinois, Chicago, IL, 60612-4319, USA
corresponding authorCorresponding author.
Jason S Buhrman: jbuhrm2/at/uic.edu; Jamie E Rayahin: jrayah2/at/uic.edu; Melanie Köllmer: mkollmer/at/uic.edu; Richard A Gemeinhart: rag/at/uic.edu
Received June 6, 2012; Accepted September 17, 2012.
Abstract
Background
Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST)-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins.
Results
Glutathione was conjugated to low molecular weight poly(ethylene glycol) diacrylate (PEGDA) via thiol-ene “click” chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH) homogenates, we were able to purify a glutathione S-transferase (GST) green fluorescent protein (GFP) fusion protein (GST-GFP) from the soluble fraction of E. coli lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads.
Conclusions
GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.
Keywords: Glutathione, PEGDA, Glutathione S-transferase, Batch purification, Recombinant protein
Articles from BMC Biotechnology are provided here courtesy of
BioMed Central