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Updated: Jan 25, 2026

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography
Published on: March 30, 2022
Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase
Sanjukta Aich1, Fumie Imabayashi, Louis T J Delbaere
1Department of Biochemistry, University of Saskatchewan, Saskatoon, Sask, Canada S7N 5E5.
Abstract:
The Corynebacterium glutamicum (C. glutamicum) phosphoenolpyruvate carboxykinase (PCK) gene (pckA) was cloned into an Escherichia coli expression vector with a glutathione S-transferase (GST) tag. This recombinant DNA can produce highly overexpressed tagged protein in soluble form. This is the first report of the production of C. glutamicum PCK overexpressed in E. coli. The GST-fused PCK was purified using the glutathione-Sepharose 4B affinity column and the GST tag was removed in one-step. This one-step, easy purification method would be very useful for future mutational and structural studies. The molecular mass of the purified protein is approximately 68 kDa as confirmed by mass spectrometry and it is a monomeric enzyme. Also, the enzyme assays revealed that C. glutamicum PCK has a GTP-specific activity and that its activity is maximal in the presence of both Mn2+ and Mg2+.
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