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Published on: July 18, 2011
Optimizing purification process of MIM-I-BAR domain by introducing atomic force microscope and dynamics simulations
Yue Zhang1, Zhichao Lou2, Xubo Lin3
1State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science & Medical Engineering, Southeast University, Nanjing, 210096, China; Collaborative Innovation Center of Suzhou Nano Science and Technology, Suzhou 215123, China.
Abstract:
MIM (missing in metastasis) is a member of I-BAR (inverse BAR) domain protein family, which functions as a putative metastasis suppressor. However, methods of gaining high purity MIM-I-BAR protein are barely reported. Here, by optimizing the purification process including changing the conditions of cell lysate and protein elution, we successfully purified MIM protein. The purity of the obtained protein was up to ∼90%. High-resolution atomic force microscope (AFM) provides more visual images, ensuring that we can observe the microenvironment around the target protein, as well as the conformations of the purification products following each purification process. MIM protein with two different sizes were observed on mica surface with AFM. Combining with molecular dynamics simulations, these molecules were revealed as MIM monomer and dimer. Furthermore, our study attaches importance to the usage of imidazole with suitable concentrations during the affinity chromatography process, as well as the removal of excessive imidazole after the affinity chromatography process. All these results indicate that the method described here was successful in purifying MIM protein and maintaining their natural properties, and is supposed to be used to purify other proteins with low solubility.
Insights
Researchers developed a new method to purify the metastasis suppressor MIM (missing in metastasis) protein. This technique achieves high purity and preserves the protein
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Purification
Background:
- The Missing in Metastasis (MIM) protein, an I-BAR domain protein, is implicated as a metastasis suppressor.
- Efficient purification methods for high-purity MIM-I-BAR protein are not well-established.
Purpose of the Study:
- To optimize and establish a reliable method for purifying MIM protein with high purity.
- To characterize the purified MIM protein using biophysical techniques.
Main Methods:
- Optimization of cell lysate conditions and protein elution steps for MIM purification.
- High-resolution atomic force microscopy (AFM) for visualizing protein conformation and microenvironment.
- Molecular dynamics simulations to analyze protein structures observed via AFM.
- Affinity chromatography utilizing imidazole for MIM protein purification.
Main Results:
- Successfully purified MIM protein to approximately 90% purity.
- AFM imaging revealed MIM monomers and dimers on mica surfaces.
- Molecular dynamics simulations confirmed the observed MIM monomer and dimer structures.
- Optimized imidazole concentration and removal during affinity chromatography were crucial.
Conclusions:
- The developed method effectively purifies MIM protein while maintaining its native properties.
- This purification strategy is potentially applicable to other low-solubility proteins.
