Related Experiment Video
Updated: Apr 20, 2026
Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers
Published on: December 6, 2019
Recombinant expression, purification and preliminary characterization of the mRNA export factor MEX67 of
Gaurav Chhetri1, Tripti Pandey1, Bijay Kumar1
1Molecular Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India.
Abstract:
The nuclear export of macromolecules is facilitated by the nuclear pore complexes (NPCs), embedded in the nuclear envelope and consists of multi-protein complexes. MEX67 is one of the nuclear export factor responsible for the transport of the majority of cellular mRNAs from the nucleus to the cytoplasm. The mechanism of mRNA transport through NPCs is unclear due to the unavailability of structures and the known interacting partners of MEX67. The mex67 gene was cloned in pQE30A and was expressed in Escherichia coli. A strategy has been developed to purify the insoluble MEX67 using a nickel affinity column with chelating Sepharose fast flow media, after solubilizing with sodium lauroyl sarcosinate (Sarkosyl). The IMAC purified recombinant MEX67 was further purified using SEC to apparent homogeneity (∼8 mg/L). Following SEC, MEX67 was stable and observed to be a 67 kDa monomeric protein as determined by PAGE and the size exclusion chromatography. The availability of large quantities of the protein will help in its biochemical and biophysical characterization, which may lead to the identification of new interaction partners of MEX67 or MEX67 complex.
Insights
Researchers purified MEX67, a key protein for mRNA export via nuclear pore complexes (NPCs). This provides ample protein for further studies on its function and interactions.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nuclear pore complexes (NPCs) mediate macromolecule transport between the nucleus and cytoplasm.
- MEX67 is a crucial nuclear export factor for messenger RNA (mRNA) transport.
- Understanding MEX67's mechanism is hindered by a lack of structural and interaction data.
Purpose of the Study:
- To develop a purification strategy for recombinant MEX67 protein.
- To obtain sufficient quantities of purified MEX67 for biochemical and biophysical characterization.
- To facilitate the identification of MEX67's interaction partners and complex.
Main Methods:
- Cloning of the mex67 gene into pQE30A expression vector.
- Expression of recombinant MEX67 in Escherichia coli.
- Purification using nickel affinity chromatography (IMAC) after solubilization with sodium lauroyl sarcosinate (Sarkosyl).
- Further purification to homogeneity using size exclusion chromatography (SEC).
Main Results:
- A robust strategy was established to purify insoluble MEX67.
- Recombinant MEX67 was purified to apparent homogeneity with a yield of approximately 8 mg/L.
- Purified MEX67 was stable, monomeric, and approximately 67 kDa, confirmed by PAGE and SEC.
- Sufficient quantities of pure MEX67 are now available for further characterization.
Conclusions:
- The developed purification protocol enables large-scale production of functional MEX67.
- Availability of purified MEX67 will aid in elucidating its role in mRNA nuclear export.
- This work lays the foundation for future studies on MEX67 interactions and its role in NPC transport.
