title: Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine.
abstract: Expression of green fluorescent protein (GFP) in Escherichia coli (E. coli) resulted in only small amount of soluble and fluorescent GFP protein and hence most of the protein in insoluble particles. The expressed GFP in insoluble particles, however, was fluorescent, indicating that it is at least in part folded with an intact chromophore. The GFP in insoluble particles could not be solubilized by an aqueous (denaturant-free) buffer. Solubilization of active GFP from insoluble particles was then attempted with guanidine hydrochloride (GdnHCl), a strong protein-denaturant, or L-arginine, an aggregation suppressor. Solubilization from insoluble particles by 6M GdnHCl led to complete denaturation of the GFP existing in insoluble particles, while GdnHCl solution at lower concentration could solubilize fluorescent GFP. Solubilization of fluorescently active GFP from insoluble particles was also achieved by L-arginine. It is noteworthy that L-arginine was stronger in solubilizing insoluble GFP than GdnHCl below 2M. These results demonstrate that some proteins expressed in E. coli may form insoluble particles containing native conformation and L-arginine may be used to recover the proteins in the native form from such insoluble particles.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 07, Aoba-ku, 980-8579, Sendai, Japan.
authors.forename: Kouhei
authors.initials: K
authors.lastname: Tsumoto
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Umetsu
authors.forename: Izumi
authors.initials: I
authors.lastname: Kumagai
authors.forename: Daisuke
authors.initials: D
authors.lastname: Ejima
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Arakawa
mesh_terms: D001120:Arginine / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D019791:Guanidine / Q000737:chemistry*; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010316:Particle Size; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D001120:Arginine; D019791:Guanidine