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Expression and purification of soluble and inactive mutant forms of membrane type 1 matrix metalloproteinase
1Department of Virology, The Haartman Institute, Helsinki, FIN-00014, Finland.
Abstract:
Membrane type 1 matrix metalloproteinase (MT1-MMP) is a membrane-bound proteinase and a cell-surface receptor and activator of gelatinase A in normal and neoplastic cells. We have expressed and purified a soluble deletion mutant of MT1-MMP lacking the transmembrane and cytoplasmic domains and an inactive mutant of the soluble MT1-MMP, where the active-site glutamic acid(240) was substituted by alanine (E240A). A baculovirus transfer vector coding for amino acids 21-539 of MT1-MMP (DeltaTM) and a similar vector coding for the mutation (E240ADeltaTM) were constructed for expression in insect cells. Both DeltaTM and E240ADeltaTM were secreted to the culture medium of infected High Five insect cells. They were then purified by cation-exchange followed by gel-filtration chromatography. DeltaTM was able to cleave denatured type I collagen and fibronectin and activate MMP-2/gelatinase-A, while E240ADeltaTM had only low proteolytic activity against denatured collagen I. The current expression and purification protocol should prove useful for the production of large amounts of enzymatically active soluble MT1-MMP.
Insights
Researchers produced soluble membrane type 1 matrix metalloproteinase (MT1-MMP) and an inactive mutant. The active soluble MT1-MMP cleaved collagen and activated MMP-2, showing its utility for further research.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Membrane type 1 matrix metalloproteinase (MT1-MMP) is crucial for extracellular matrix remodeling.
- MT1-MMP functions as a protease and an activator of MMP-2 (gelatinase A).
- Understanding MT1-MMP's enzymatic activity requires soluble, active forms for study.
Purpose of the Study:
- To express and purify a soluble deletion mutant of MT1-MMP (DeltaTM).
- To create and characterize an enzymatically inactive mutant (E240ADeltaTM).
- To establish a protocol for producing large quantities of active soluble MT1-MMP.
Main Methods:
- Constructed baculovirus transfer vectors for soluble (DeltaTM) and inactive (E240ADeltaTM) MT1-MMP mutants.
- Expressed mutants in High Five insect cells using a baculovirus system.
- Purified proteins via cation-exchange and gel-filtration chromatography.
Main Results:
- Both DeltaTM and E240ADeltaTM were secreted and purified.
- Soluble DeltaTM demonstrated proteolytic activity against denatured type I collagen and fibronectin.
- DeltaTM effectively activated MMP-2, while E240ADeltaTM showed minimal collagenolytic activity.
Conclusions:
- The developed expression and purification protocol yields significant amounts of active soluble MT1-MMP.
- This method facilitates further investigation into MT1-MMP's biochemical functions.
- The study provides a valuable tool for studying MT1-MMP in various biological contexts.