title: Cloning, expression and purification of functionally active human angiopoietin-like protein 2.
abstract: Angiopoietin-like protein 2 (Angptl2) is a secreted glycoprotein that has been implicated in angiogenesis, inflammation and atherosclerosis as well as enhancing the survival of human hematopoietic stem cells. Glycosylation of Angptl2 is required for biological activity and studies of angiopoietin-like protein 2 have been hindered by the lack of a source for the mature form of this protein. We describe a system that permits purification of the glycosylated form of human Angptl2 from conditioned media of stably transfected HEK 293 cells. To facilitate purification while retaining the integrity of Angptl2's endogenous N-terminal secretion signal peptide, GST was fused downstream of the Angptl2 coding sequence. Secreted Angptl2-GST was purified using a one-step glutathione-affinity purification scheme. The purity and identity of the resulting protein were confirmed by SDS-PAGE, immunoblotting, and mass spectrometry. N-Glycosidase treatment reduced the apparent molecular mass of Angptl2-GST on SDS-PAGE, confirming its glycosylation state. Purified human Angptl2-GST stimulated both HUVEC migration and microtubule formation in vitro. The yield of Angptl2-GST obtained was in quantities suitable for multiple applications including functional in vitro and in vivo assays.
authors: Departments of Pharmacology, Montreal, QC H3T 1J4 Canada ; Montreal Heart Institute, Montréal, Québec H1T 1C8 Canada ; Pharsight Corporation Canada, 2000 Peel, Montreal, Québec H3A 2W5 Canada.
authors: Nada
authors: N
authors: Farhat
authors: Montreal Heart Institute, Montréal, Québec H1T 1C8 Canada.
authors: Aida M
authors: AM
authors: Mamarbachi
authors: Departments of Pharmacology, Montreal, QC H3T 1J4 Canada ; Department of Surgery, Université de Montréal, Université de Montréal, 5000 Belanger St, Montréal, Québec H1T 1C8 Canada ; Montreal Heart Institute, Montréal, Québec H1T 1C8 Canada.
authors: Eric
authors: E
authors: Thorin
authors: Departments of Biochemistry, Montreal, QC H3T 1J4 Canada ; Department of Medicine, Montreal Heart Institute, Université de Montréal, 5000 Belanger St, Montréal, Québec H1T 1C8 Canada ; Montreal Heart Institute, Montréal, Québec H1T 1C8 Canada ; Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6 Canada.
authors: Bruce G
authors: BG
authors: Allen
keywords: Angiogenesis; Angiopoietin-like protein 2; Atherosclerosis; Glycoprotein; HEK 293 cells; Protein expression; Purification