A new protein folding screen: application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase.
Production of folded and biologically active protein from Escherichia coli derived inclusion bodies can only be accomplished if a scheme exists for in vitro naturation. Motivated by the need for a rapid and statistically meaningful method of determining and evaluating protein folding conditions, we have designed a new fractional factorial protein folding screen. The screen includes 12 factors shown by previous experiments to enhance protein folding and it incorporates the 12 factors into 16 different folding conditions. By examining a 1/256th fraction of the full factorial, multiple folding conditions were determined for the ligand binding domains from glutamate and kainate receptors, and for lysozyme and carbonic anhydrase B. The impact of each factor on the formation of biologically active material was estimated by calculating factor main effects. Factors and corresponding levels such as pH (8.5) and L-arginine (0.5 M) consistently had a positive effect on protein folding, whereas detergent (0.3 mM lauryl maltoside) and nonpolar additive (0.4 M sucrose) were detrimental to the folding of these four proteins. One of the 16 conditions yielded the most folded material for three out of the four proteins. Our results suggest that this protein folding screen will be generally useful in determining whether other proteins will fold in vitro and, if so, what factors are important. Furthermore, fractional factorial folding screens are well suited to the evaluation of previously untested factors on protein folding.
Armstrong
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N
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.
de Lencastre
A
A
Gouaux
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E
D000595:Amino Acid Sequence; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000378:metabolism*; D017931:DNA Primers; D008024:Ligands; D009113:Muramidase / Q000378:metabolism*; D011485:Protein Binding; D017470:Receptors, Glutamate / Q000378:metabolism*; D018092:Kainic Acid Receptors / Q000378:metabolism*
D002256:Carbonic Anhydrases; D017931:DNA Primers; D008024:Ligands; D009113:Muramidase; D017470:Receptors, Glutamate; D018092:Kainic Acid Receptors