Production and Biochemical Characterization of Dimeric Recombinant Gremlin-1.

Stefania Mitola,Alessandra Gianoncelli,Kurt Ballmer-Hofer,Michela Corsini,Cosetta Ravelli,Federico Galvagni,Marco Presta,Elisabetta Grillo

doi:10.3390/ijms23031151

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.

Highlights

The gremlin-1 gene is highly conserved across evolution and, in humans, it encodes for a 184 amino acid long protein, whose theoretical molecular weight is 20.682 Da [1].Gremlin-1 is a highly basic polypeptide, containing 7.61% arginine, 8.7% lysine and 2.17% histidine residues
Gremlin is found in the endoplasmic reticulum and associated with the cell surface, where it binds to heparan sulfate proteoglycans (HSPGs) once it has been secreted [2]
Such studies benefited from the availability of a recombinant form of gremlin that has contributed to rapid advancements in gremlin-related research

Summary

Introduction

The gremlin-1 gene is highly conserved across evolution and, in humans, it encodes for a 184 amino acid long protein, whose theoretical molecular weight is 20.682 Da [1]. Gremlin-1 (hereinafter referred to as gremlin) is a highly basic polypeptide (pI = 9.53), containing 7.61% arginine, 8.7% lysine and 2.17% histidine residues. Gremlin is found in the endoplasmic reticulum and associated with the cell surface, where it binds to heparan sulfate proteoglycans (HSPGs) once it has been secreted [2]. Gremlin contains three serine residues, which may undergo phosphorylation (Ser, Ser140 and Ser142). Ser was confirmed to be phosphorylated [3]. Gremlin is N-glycosylated on Asn42 [2]. The biological role of such modifications is not known

Methods

Results

Conclusion