An automatable screen for the rapid identification of proteins amenable to refolding.
Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is hampered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under non-denaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an individual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous sample preparation requirements. The efficacy of the screen is demonstrated on small-scale expression samples for 15 proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism.
Cowieson
Nathan P
NP
Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane, Queensland, Australia. n.cowieson@imb.uq.edu.au
Wensley
Beth
B
Listwan
Pawel
P
Hume
David A
DA
Kobe
Bostjan
B
Martin
Jennifer L
JL
D000818:Animals; D019149:Bioreactors / Q000382:microbiology; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003720:Densitometry; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D023281:Genomics; D006639:Histidine / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D002479:Inclusion Bodies / Q000737:chemistry; D051379:Mice; D008970:Molecular Weight; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012995:Solubility; D014508:Urea / Q000494:pharmacology
D011506:Proteins; D020543:Proteome; D006639:Histidine; D014508:Urea