title: Expression, purification, and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli.
abstract: Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni(2+) affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe(X) (K(d) = 321 nM, B(max) = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.
authors.affiliation: School of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, 061-0293, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Kawano
authors.forename: Daisuke
authors.initials: D
authors.lastname: Iyaguchi
authors.forename: Chiaki
authors.initials: C
authors.lastname: Okada
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Eiko
authors.initials: E
authors.lastname: Toyota
mesh_terms: D002846:Chromatography, Affinity; D019040:E-Selectin / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D019040:E-Selectin; C546936:SELE protein, human; D004815:Epidermal Growth Factor