Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells

Lena Vinther, Ulrik Lademann, Elisabeth Veyhe Andersen, Peter Højrup, Morten Thaysen-Andersen, Berit Olsen Krogh, Birgitte Viuff, Nils Brünner, Jan Stenvang, José M A Moreira

Research output: Contribution to journalJournal articleResearchpeer-review

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.

Original languageEnglish
JournalProtein Expression and Purification
Volume101
Pages (from-to)157-64
Number of pages8
ISSN1046-5928
DOIs
Publication statusPublished - Sep 2014

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Tissue Inhibitor of Metalloproteinase-1
Proteins
Glycosylation
Matrix Metalloproteinase Inhibitors
HEK293 Cells
Matrix Metalloproteinase 2
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Growth
Matrix Metalloproteinases
human TIMP1 protein
Cell Line
Neoplasms

Cite this

Vinther, Lena ; Lademann, Ulrik ; Andersen, Elisabeth Veyhe ; Højrup, Peter ; Thaysen-Andersen, Morten ; Krogh, Berit Olsen ; Viuff, Birgitte ; Brünner, Nils ; Stenvang, Jan ; Moreira, José M A. / Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells. In: Protein Expression and Purification. 2014 ; Vol. 101. pp. 157-64.
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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",
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.",
author = "Lena Vinther and Ulrik Lademann and Andersen, {Elisabeth Veyhe} and Peter H{\o}jrup and Morten Thaysen-Andersen and Krogh, {Berit Olsen} and Birgitte Viuff and Nils Br{\"u}nner and Jan Stenvang and Moreira, {Jos{\'e} M A}",
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year = "2014",
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language = "English",
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pages = "157--64",
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Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells. / Vinther, Lena; Lademann, Ulrik; Andersen, Elisabeth Veyhe; Højrup, Peter; Thaysen-Andersen, Morten; Krogh, Berit Olsen; Viuff, Birgitte; Brünner, Nils; Stenvang, Jan; Moreira, José M A.

In: Protein Expression and Purification, Vol. 101, 09.2014, p. 157-64.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Purification and characterization of bioactive his6-tagged recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) protein expressed at high yields in mammalian cells

AU - Vinther, Lena

AU - Lademann, Ulrik

AU - Andersen, Elisabeth Veyhe

AU - Højrup, Peter

AU - Thaysen-Andersen, Morten

AU - Krogh, Berit Olsen

AU - Viuff, Birgitte

AU - Brünner, Nils

AU - Stenvang, Jan

AU - Moreira, José M A

N1 - Copyright © 2014 Elsevier Inc. All rights reserved.

PY - 2014/9

Y1 - 2014/9

N2 - 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.

AB - 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.

U2 - 10.1016/j.pep.2014.06.013

DO - 10.1016/j.pep.2014.06.013

M3 - Journal article

C2 - 24998777

VL - 101

SP - 157

EP - 164

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

ER -