High-throughput identification of refolding conditions for LXRbeta without a functional assay.
The expression of human genes in bacteria is often one of the most efficient systems for generating proteins for drug discovery efforts. However, expression of mammalian cDNAs in Escherichia coli often results in the production of protein that is insoluble and misfolded and thus requires the development of a successful refolding procedure to generate active protein. To accelerate the process of developing protein refolding protocols, we have developed a semi-automated screening and assay system that utilizes an incomplete factorial approach to sample a large "space" of refolding conditions based on parameters known to influence protein stability and solubility. Testing of these conditions is performed readily in a 96-well plate format with minimal sample manipulation. The folded protein is resolved and detected using an HPLC equipped with a mini-column and a highly sensitive fluorescence detector. This simple method requires only a small amount of protein for the entire screen (<1 mg), and most importantly, a functional assay is not required to assess the refolding yields. Here, we validate the utility of this screening system using two model proteins, IL13 and MMP13, and demonstrate its successful application to the refolding of our target protein, the ligand-binding domain of rat liver X receptor beta.
Lin
Laura
L
Department of Structural Biology and Computational Chemistry, Chemical and Screening Sciences, Wyeth Research, Cambridge, MA 02140, USA. lllin@wyeth.com
Seehra
Jasbir
J
Stahl
Mark L
ML
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D017364:Collagenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D018793:Interleukin-13 / Q000737:chemistry* / Q000191:economics / Q000378:metabolism; D000071518:Liver X Receptors; D053509:Matrix Metalloproteinase 13; D057093:Orphan Nuclear Receptors; D017510:Protein Folding; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D004268:DNA-Binding Proteins; D018793:Interleukin-13; D000071518:Liver X Receptors; D057093:Orphan Nuclear Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; C507083:Mmp13 protein, rat