The ORF1p protein encoded by the LINE1 retrotransposon, the only active autonomous retrotransposon in the human genome, is an approximately 40 kDa RNA-binding protein with nucleic acid chaperone activity. In recent years, it has gained increasing attention in research related to neurodegenerative diseases. Our aim was to express and isolate ORF1p in sufficient quantity for further experiments, such as a nuclear import assay. We constructed the vectors pMCSG7-MBP-ORF1p (N-terminal fusion with MBP and His6) for the expression of MBP-ORF1p and pMCSG7-ORF1p-MBP (C-terminal fusion with MBP and His6) for the expression of ORF1p-MBP. The His6 tag enabled protein purification by immobilized metal affinity chromatograph (IMAC), while the MBP tag allowed purification by MBP affinity chromatography on a dextrin-sepharose column. The ORF1p-MBP construct exhibited a higher degree of degradation during expression and purification compared to MBP-ORF1p. MBP-ORF1p was isolated with comparable purity using both purification methods. In both cases, a substantial amount of degradation products was detected in the elution fractions, as confirmed by immunodetection. We concluded that protein degradation was likely due to the presence of intrinsically disordered regions in ORF1p, which are more susceptible to proteolysis. The isolation procedure was partially improved by the addition of glycerol to the binding and elution buffers. Further optimization of the protocol and reduction of protease activity would be necessary. The His6-MBP tag was successfully removed from MBP-ORF1p using TEV protease.
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