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Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.14/341060
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- 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
- Related
- Protein expression and purification, Vol. 101, (2014), p.157-164
- DOI
- 10.1016/j.pep.2014.06.013
- Publisher
- Elsevier
- Date
- 2014
- Author/Creator
- Vinther, Lena
- Author/Creator
- Lademann, Ulrik
- Author/Creator
- Andersen, Elisabeth Veyhe
- Author/Creator
- Højrup, Peter
- Author/Creator
- Thaysen-Andersen, Morten
- Author/Creator
- Krogh, Berit Olsen
- Author/Creator
- Viuff, Birgitte
- Author/Creator
- Brünner, Nils
- Author/Creator
- Stenvang, Jan
- Author/Creator
- Moreira, José M. A
- Description
- Tissue inhibitor of metalloproteinase-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.
- Description
- 8 page(s)
- Subject Keyword
- Tissue inhibitor of metalloproteinase-1 protein
- Subject Keyword
- His tagged
- Subject Keyword
- Bioactive
- Subject Keyword
- HEK293E expression system
- Resource Type
- journal article
- Organisation
- Macquarie University. Department of Chemistry and Biomolecular Sciences
- Identifier
- http://hdl.handle.net/1959.14/341060
- Identifier
- mq:37876
- Identifier
- ISSN:1046-5928
- Identifier
- mq-rm-2013010778
- Identifier
- mq_res-ext-20150416-1548-10
- Language
- eng
- Reviewed
Macquarie University ResearchOnline
