Expression and characterization of E-LecEGF for structural study and assay development

Human E-selectin (hE-selectin) is a cell adhesion molecule expressed on the membrane of endothelial cells. It is a C-type lectin whose key role is to mediate the initial rolling and adhering of leukocytes in the leukocyte recruitment in inflammation and metastasis of some cancer cells. It is fundamentally involved in many physiological and pathological processes, and hence is an attractive target for developing anti-inflammation drugs. The lectin and EGF domains of hE-selectin (hE-LecEGF) were identified as the minimum functional unit. Crystal structures of hE-LecEGF complexed with its natural ligand, tetrasaccharide sialyl Lewisx (sLex), as well as NMR studies of hE-selectin/IgG bound with this ligand, have been reported and utilized as the structural basis for the development of potent antagonists for hE-selectin. More potent antagonists with better binding affinity than sLex, such as CGP69669, were reported, but their binding modes in hE-LecEGF remain unknown. To obtain the improved structural information of hE-LecEGF complexed with an antagonist and develop more potent antagonists of hE-selectin are challenging tasks. To meet the demands of the protein for the structure determination and the binding assay, a sufficient amount of pure and active hE-LecEGF is needed. In this thesis, insect cell expression systems were initially used to produce the hE-LecEGF protein. hE-LecEGF was cloned, transiently expressed and characterized in Sf9 and High fiveTM cells. The expression plasmid pFastBacYJSE was constructed for expression of the hE-LecEGF protein fused with a N-terminal Flag tag. The recombinant baculovirus was generated and used in the expression of protein in the suspension culture. Pure hE-LecEGF was obtained by anti-Flag M2 affinity chromatography under the optimized condition. The purified protein was active and glycosylated, as identified by mAb 7A9 and glycan detection, respectively. Unfortunately, the homogeneous hE-LecEGF protein was not obtained after the deglycosylation with PNGase F and N-glycosidase A. hE-LecEGF was then cloned, stably expressed and characterized in CHO K1 cells. Stable subclones CHO-YJES and CHO-YJEGS expressing the hE-LecEGF protein with or without a human IgG1 tag were achieved. The CHO-YJES construct was used for production. The monoclonal anti-E-selectin functional blocking antibody 7A9 (mAb 7A9) was produced, purified and coupled to sepharose for functional purification of the hE-LecEGF protein. Highly pure hE-LecEGF protein was obtained in a one-step purification with an mAb 7A9 coupled column. Page, western-blotting, ELISA, MS and NMR were performed to characterize the hE-LecEGF protein. Pure, monomeric and active hE-LecEGF with the molecular weight of 20.444 kDa was obtained. In contrast to the insect cell expression system, pure, active and
uniform deglycosylated hE-LecEGF protein was obtained after treatment with PNGase F and purification by a Sepharose Q matrix. A prescreening of the crystallization condition of hE-LecEGF was also performed using a sitting-drop method. Furthermore, a novel cell-free assay “capture-binding assay” was developed with the tag-free hE-LecEGF protein to evaluate the binding activity of the hE-LecEGF protein and the binding affinity of hE-selectin antagonists. The rIC50 of six hE-selectin antagonists was determined. The obtained results were in close agreement with the published results. Compared to the previously unstable polymer assay with hE-selectin/IgG, the capture-binding assay with hE-LecEGF is accurate, sensitive and reproducible. It can correctly evaluate the binding affinities of hE-selectin antagonists. In addition, the antibody BBA1 was used to solve the problem of immobilization of the hE-LecEGF protein on ELISA plates in the assay.