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.

Functional Characterization of a Conserved Archaeal Viral Operon Revealing Single-Stranded DNA Binding, Annealing and Nuclease Activities

“…and gp18-His was purified using Ni-NTA-agarose beads. The denatured gp18-His was then refolded in L-Arginine buffer (29).…”

Molecular characterization of a fungal cyclophilin allergen Rhi o 2 and elucidation of antigenic determinants responsible for IgE–cross-reactivity

“…Recombinant proteins (TV-3 and TV-4) expressed in inclusion bodies were purified under denaturing conditions using 7 M urea buffer and then made soluble by a rapid dilution method as described in Ref. 39.…”

Expression of Lectins in Heterologous Systems

“…There are considerable differences between eukaryotic lectins synthesized in ER and those that have been synthesized in the cytoplasm. First, the ER lumen is a highly oxidizing environment that favours the formation of disulphide bonds (S–S), while the cytosol maintains the cysteine residues in their reduced form (–SH) [ 9 , 25 ]. On the other hand, the ER lumen contains accessory proteins involved in proper protein folding [ 23 ] and has the necessary proteases to cleave the lectin precursors that need to be processed to their functional form, such as in the case of ConA from Canavalia ensiformis and favina from Vicia faba [ 25 ].…”

“…Unfortunately, such products are often not soluble or not properly processed. In an attempt to circumvent these disadvantages, a number of alternatives have emerged, ranging from refolding [ 8 , 9 , 10 ], the inclusion of solubilizing peptides [ 11 , 12 ] and the modification of the microorganism glycosylation patterns [ 13 ]. Some of these modifications include the expression of human enzymes in yeast strains of Pichia pastoris , which are commercially available.…”