title: Recombinant expression, purification and preliminary characterization of the mRNA export factor MEX67 of Saccharomyces cerevisiae.
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.
authors.affiliation: Molecular Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Chhetri
authors.affiliation: Molecular Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India.
authors.forename: Tripti
authors.initials: T
authors.lastname: Pandey
authors.affiliation: Molecular Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India.
authors.forename: Bijay
authors.initials: B
authors.lastname: Kumar
authors.affiliation: Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Jankipuram Extension, Lucknow 226031, India. Electronic address: sohail@cdri.res.in.
authors.forename: Md Sohail
authors.initials: MS
authors.lastname: Akhtar
authors.affiliation: Molecular Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India. Electronic address: timir.tripathi@gmail.com.
authors.forename: Timir
authors.initials: T
authors.lastname: Tripathi
mesh_terms: D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: C106898:MEX67 protein, S cerevisiae; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins
keywords: Chromatography; Detergent; Ethanol; Expression; Purification; Solubility