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Functional recombinant rabbit muscle phosphoglucomutase from Escherichia coli.
1Department of Biochemistry, National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706, USA.
Protein Expression and Purification
|October 19, 2000
Summary
A bacterial system was developed to produce rabbit phosphoglucomutase (PGM) protein for NMR studies. The purified PGM protein is over 90% pure and suitable for stable-isotope labeling experiments.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Expression
Background:
- Phosphoglucomutase (PGM) is crucial in carbohydrate metabolism.
- Stable-isotope labeled PGM is needed for advanced Nuclear Magnetic Resonance (NMR) studies.
- Existing methods may not provide sufficient quantities of PGM for these experiments.
Purpose of the Study:
- To establish a bacterial expression system for producing rabbit phosphoglucomutase (PGM).
- To obtain highly pure, stable-isotope labeled PGM for NMR applications.
- To assess the suitability of the expressed PGM for NMR investigations.
Main Methods:
- Expressed rabbit PGM gene in Escherichia coli using a T7 expression system with a His-tag.
- Purified soluble PGM protein using a one-step Ni-NTA column chromatography.
- Assessed protein purity and activity via gel electrophoresis and enzyme assays.
Main Results:
- Successfully expressed and purified PGM protein from E. coli.
- Achieved a yield of approximately 10 mg per liter of LB medium.
- The purified PGM protein demonstrated >90% purity and activity (128 U/mg).
- Preliminary NMR studies confirmed adequate purity for planned experiments.
Conclusions:
- The developed bacterial system provides a viable method for producing rabbit PGM.
- The His-tag does not impede PGM activity and is suitable for initial NMR studies.
- Future work includes designing a cleavable His-tag for more detailed active site NMR analysis.