Escherichia coli fusion carrier proteins act as solubilizing agents for recombinant uncoupling protein 1 through interactions with GroEL.
Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein.
Douette
Pierre
P
Laboratory of Bioenergetics, Centre of Oxygen Research and Development, Institut de Chimie B6, Université de Liège, Sart Tilman, B-4000 Liege, Belgium.
Navet
Rachel
R
Gerkens
Pascal
P
Galleni
Moreno
M
Lévy
Daniel
D
Sluse
Francis E
FE
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007473:Ion Channels; D037601:Mannose-Binding Lectin / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D007473:Ion Channels; D037601:Mannose-Binding Lectin; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D000071256:Uncoupling Protein 1