Production and Biochemical Characterization of Dimeric Recombinant Gremlin-1

Abstract

Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.

Keywords: HEK293T expression system; cystine-knot protein; dimer; gremlin-1.

Figure 1

Expression and IMAC purification of recombinant gremlin in HEK293T cells. (A) WB analysis of gremlin expression in the supernatant (SN) or the lysate (Ly) of HEK293T cells transiently transfected with pcDNA3-Gremlin-1-HisTag (gremlin) or empty vector (ctrl). (B) WB analysis of gremlin expression in the supernatant of HEK293T cells in the absence or the presence of 6 mM sodium butyrate. (C) WB analysis of gremlin expressed and secreted by HEK293T cells in the absence or the presence of PNGaseF glycosidase. (D) WB analysis of gremlin expressed and secreted by HEK293T cells 0–36 h post transfection. (E–H) IMAC of recombinant gremlin expressed in the supernatant of HEK293T cells. The chromatogram is shown in (E). Fractions were analyzed by WB for gremlin presence (F), or by silver staining (G). Non-reducing SDS-PAGE followed by anti-gremlin WB of purified gremlin is shown in (H). (I) MALDI-TOF/TOF–MS spectrum of IMAC-purified recombinant gremlin. Arrows indicate the two species of gremlin that were detected. (J) Total yield of IMAC gremlin purification. Data for one representative experiment of at least 3 independent repeats are shown.

Figure 2

IMAC-purified gremlin retained full BMP-dependent activity and only mild VEGFR2 activation capacity. (A) WB analysis of phospho-SMAD1/5/8 levels in HepG2 cells stimulated with BMP4 in the presence or the absence of indicated doses of IMAC-purified gremlin. FAK, loading control. (B) WB analysis of phospho-VEGFR2 levels in HUVE cells stimulated with increasing doses of IMAC-purified gremlin. FAK, loading control. (C) HUVE cells sprouting assay in the absence or the presence of increasing doses of IMAC-purified gremlin. Data are expressed as mean ± SEM. *, p < 0.05 Student’s t test vs. untreated cells. Data from one representative experiment of at least 3 independent repeats are shown.

Figure 3

Heparin-affinity chromatography allows isolating pure dimeric recombinant gremlin. IMAC-purified gremlin was subjected to a second step of purification through heparin-affinity chromatography. (A) WB analysis under non-reducing and reducing conditions of fractions eluted from the heparin-affinity chromatography. (B) Total yield of gremlin purification through heparin-affinity chromatography. Data from one representative experiment of at least 3 independent repeats are shown.

Figure 4

Pure dimeric gremlin exhibits strong biological activity. (A) Activation of Hepcidin promoter in a luciferase reporter assay upon BMP4 stimulation (50 ng/mL). When indicated, BMPs were pre-incubated in the absence, or presence, of the indicated concentration (ng/mL) of IMAC-purified or dimeric heparin-sepharose (HepSep)-purified gremlin. (B) pVEGFR2 accumulation into the VPM of GM7373-VEGFR2 endothelial cells adherent to IMAC-purified or dimeric HepSep-purified gremlin (dishes were coated with 2 µg/mL of recombinant proteins). (C) Cell proliferation of HUVE cells stimulated with increasing doses (ng/mL) of IMAC- or HepSep-purified gremlin. Data are expressed as mean ± SEM of three independent experiments. *, p < 0.05 Student’s t test vs. BMP-treated cells. #, p < 0.05 Student’s t test vs. gremlin IMAC-treated cells.