title: Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells.
abstract: Tissue inhibitor of metalloproteinases-1 (TIMP-1) is an endogenous inhibitor of matrix metalloproteinases (MMPs) with reported tumor promoting, as well as inhibitory, effects. These paradoxical properties are presumably mediated by different biological functions, MMP-dependent as well as -independent, and probably related to TIMP-1 levels of protein expression, post-translational modifications, and cellular localization. TIMP-1 is an N-glycosylated protein that folds into two functional domains, a C- and an N-terminal domain, with six disulfide bonds. Furthermore, TIMP-1 is processed in the N-terminal sequence. These three biochemical properties make TIMP-1 difficult to produce in conventional bacterial, insect, or yeast expression systems. We describe here a HEK293 cell-based strategy for production and purification of secreted and N-glycosylated recombinant his6-tagged human TIMP-1 (his6-rTIMP-1), which resulted in large amounts of highly purified and bioactive protein. Matrix-assisted laser desorption ionization mass spectrometry confirmed the N- and C-termini of his6-rTIMP-1, and N-glycosylation profiling showed a match to the N-glycosylation of human plasma TIMP-1. The his6-rTIMP-1 was bioactive as shown by its proper inhibitory effect on MMP-2 activity, and its stimulatory effect on cell growth when added to the growth medium of four different breast cancer cell lines. This study provides an easy set-up for large scale production and purification of bioactive, tagged recombinant human TIMP-1, which structurally and functionally is similar to endogenous human TIMP-1, while using an expression system that is adaptable to most biochemical and biomedical laboratories including those that do not perform protein purifications routinely.
authors.affiliation: Section for Molecular Disease Biology and Sino-Danish Breast Cancer Research Centre, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
authors.forename: Lena
authors.initials: L
authors.lastname: Vinther
authors.affiliation: Section for Molecular Disease Biology and Sino-Danish Breast Cancer Research Centre, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
authors.forename: Ulrik
authors.initials: U
authors.lastname: Lademann
authors.affiliation: Section for Molecular Disease Biology and Sino-Danish Breast Cancer Research Centre, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
authors.forename: Elisabeth Veyhe
authors.initials: EV
authors.lastname: Andersen
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Højrup
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Thaysen-Andersen
authors.affiliation: Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
authors.forename: Berit Olsen
authors.initials: BO
authors.lastname: Krogh
authors.affiliation: Section for Molecular Disease Biology and Sino-Danish Breast Cancer Research Centre, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
authors.forename: Birgitte
authors.initials: B
authors.lastname: Viuff
authors.affiliation: Section for Molecular Disease Biology and Sino-Danish Breast Cancer Research Centre, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
authors.forename: Nils
authors.initials: N
authors.lastname: Brünner
authors.affiliation: Section for Molecular Disease Biology and Sino-Danish Breast Cancer Research Centre, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
authors.forename: Jan
authors.initials: J
authors.lastname: Stenvang
authors.affiliation: Section for Molecular Disease Biology and Sino-Danish Breast Cancer Research Centre, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark. Electronic address: jomo@sund.ku.dk.
authors.forename: José M A
authors.initials: JM
authors.lastname: Moreira
mesh_terms: D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000188:drug therapy; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D061986:MCF-7 Cells; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology*
chemical_list: D061965:Matrix Metalloproteinase Inhibitors; D011994:Recombinant Proteins; C560822:TIMP1 protein, human; D019715:Tissue Inhibitor of Metalloproteinase-1; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2
keywords: Bioactive; HEK293E expression system; His tagged; Tissue inhibitor of metalloproteinase-1 protein