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Protein J (2013) 32:386–391 DOI 10.1007/s10930-013-9496-z Expression, Puri?cation, and Refolding of Active Recombinant Human E-selectin Lectin and EGF Domains in Escherichia coli Susumu Kawano ? Daisuke Iyaguchi ? Chiaki Okada ? Yusuke Sasaki ? Eiko Toyota Published online: 29 May 2013 ó Springer Science+Business Media New York 2013 Abstract Attempts to obtain active E-selectin from sLex-PAA-Fluor Fluorescence-labeled sialyl Lewis X Escherichia coli (E. coli) have not yet been successful. In polyacrylamide this study, we succeeded in expressing the recombinant GFP Green ?uorescent protein lectin and epidermal growth factor domain fragments of L-Arg L-Arginine human E-selectin (rh-ESLE) in E. coli on a large-scale. The IPTG b-D-1-thiogalactopyranoside rh-ESLE protein was expressed as an inactive form in the rh-ESLE Recombinant human E-selectin lectin inclusion bodies. The inactive form of rh-ESLE was and epidermal growth factor domain denatured and solubilized by 6 M guanidine hydrochloride fragment and then puri?ed by Ni2? af?nity chromatography under Gua Guanidine hydrochloride denaturing conditions. Denatured rh-ESLE was then DTT Dithiothreitol refolded by a rapid-dilution method using a large amount PBS ? Calcium and magnesium containing of refolding buffer, which contained arginine and cysteine/ phosphate buffer saline cystine. The refolded rh-ESLE showed binding af?nity for PBS - Calcium and magnesium free phosphate X sLe (Kd = 321 nM, Bmax = 1.9 pmol/lg protein). This buffer saline result suggests that the refolded rh-ESLE recovered its Kd Binding constant native and functional structure. CHO Chinese hamster ovary Keywords Recombinant human E-selectin á Rapid-dilution refolding method á Binding assay á 1 Introduction E. coli á Arginine