title: Visualization of coupled protein folding and binding in bacteria and purification of the heterodimeric complex.
abstract: During overexpression of recombinant proteins in Escherichia coli, misfolded proteins often aggregate and form inclusion bodies. If an aggregation-prone recombinant protein is fused upstream (as an N-terminal fusion) to GFP, aggregation of the recombinant protein domain also leads to misfolding of the downstream GFP domain, resulting in a decrease or loss of fluorescence. We investigated whether the GFP domain could fold correctly if aggregation of the upstream protein domain was prevented in vivo by a coupled protein folding and binding interaction. Such interaction has been previously shown to occur between the E. coli integration host factors alpha and beta, and between the domains of the general transcriptional coactivator cAMP response element binding protein (CREB)-binding protein and the activator for thyroid hormone and retinoid receptors. In this study, fusion of integration host factor beta or the CREB-binding protein domain upstream to GFP resulted in aggregation of the fusion protein. Coexpression of their respective partners, on the other hand, allowed soluble expression of the fusion protein and a dramatic increase in fluorescence. The study demonstrated that coupled protein folding and binding could be correlated to GFP fluorescence. A modified miniintein containing an affinity tag was inserted between the upstream protein domain and GFP to allow rapid purification and identification of the heterodimeric complex. The GFP coexpression fusion system may be used to identify novel protein-protein interactions that involve coupled folding and binding or protein partners that can solubilize aggregation-prone recombinant proteins.
authors.affiliation: New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA.
authors.forename: Haoyong
authors.initials: H
authors.lastname: Wang
authors.forename: Shaorong
authors.initials: S
authors.lastname: Chong
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D034982:Integration Host Factors / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
chemical_list: D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D034982:Integration Host Factors; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C020723:citrate-binding transport protein; C440502:integration host factor, E coli; D049452:Green Fluorescent Proteins