Related Experiment Video
Updated: May 9, 2026
Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography
Published on: March 30, 2022
Efficient soluble expression of secreted matrix metalloproteinase 26 in Brevibacillus choshinensis
Tianyang Mu1, Weiguo Liang, Ying Ju
1Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, School of Life Sciences, Jilin University, 2699 Qianjin Street, Changchun, Jilin 130012, PR China.
Abstract:
Matrix metalloproteinase 26 (MMP-26) is a novel member of the matrix metalloproteinase family with minimal domain constitution and unknown physiological function. The three-dimensional (3D) structure of the enzyme also remains to be deciphered. Previous studies show that MMP-26 may be expressed in Escherichia coli (E. coli) as inclusion bodies and re-natured with catalytic activity. However, the low re-naturation rate of this method limits its usage in structural studies. In this paper, we tried to clone, express and purify the pro form and catalytic form of MMP-26 (ProMMP-26 and CatMMP-26) in several widely used expression vectors and express the recombinant MMP-26 proteins in E. coli cells. These constructs resulted in insoluble expressions or soluble expressions of MMP-26 with little catalytic activity. We then used Brevibacillus choshinensis (B. choshinensis) as the host system for the soluble and active expression of MMP-26. The enzyme was secreted in soluble form in the supernatant of cell culture medium and purified via a two-step purification process that included Ni(2+) affinity chromatography followed by gel filtration. The yields of purified ProMMP-26 and CatMMP-26 were 12 and 18mg/L, respectively, with high purity and homogeneity. Both ProMMP-26 and CatMMP-26 showed gelatin zymography activity and the purified CatMMP-26 had high enzymatic activity against DQ-gelatin substrate. The large-scale soluble and active protein production for future structural studies of MMP-26 is thus feasible using the B. choshinensis host system.
Insights
Matrix metalloproteinase 26 (MMP-26) was successfully expressed in Brevibacillus choshinensis, yielding soluble and active forms. This breakthrough enables large-scale production for future structural and functional studies of MMP-26.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Matrix metalloproteinase 26 (MMP-26) is a novel matrix metalloproteinase with an incompletely understood physiological role and undetermined 3D structure.
- Previous attempts to express MMP-26 in Escherichia coli resulted in inclusion bodies or low catalytic activity after refolding, hindering structural investigations.
Purpose of the Study:
- To develop a method for soluble and active expression of matrix metalloproteinase 26 (MMP-26) for future structural studies.
- To compare the expression of MMP-26 in Escherichia coli versus Brevibacillus choshinensis.
Main Methods:
- Cloning and expression of pro-MMP-26 and catalytic MMP-26 (CatMMP-26) in E. coli using various vectors, leading to insoluble or poorly active protein.
- Utilizing Brevibacillus choshinensis as a host system for soluble and secreted expression of MMP-26.
- Purification of recombinant MMP-26 using Ni(2+) affinity chromatography and gel filtration.
Main Results:
- Expression in E. coli yielded insoluble protein or soluble forms with minimal activity.
- Brevibacillus choshinensis facilitated high-yield (12-18 mg/L) soluble and active secretion of ProMMP-26 and CatMMP-26.
- Purified CatMMP-26 exhibited significant enzymatic activity against DQ-gelatin substrate.
Conclusions:
- Brevibacillus choshinensis is an effective host system for large-scale, soluble, and active production of MMP-26.
- The developed method provides a feasible approach for obtaining sufficient MMP-26 for detailed structural and functional analyses.
- This work overcomes previous limitations in MMP-26 expression, paving the way for its structural elucidation.
