Splicing factor proline and glutamine rich (SFPQ) is a protein that interacts with protein,
RNA and DNA. Because of that, it has a plethora of cell functions and is increasingly
popular among neurodegenerative disease studies. Our aim was to express and purify
enough SFPQ to conduct experiments such as monitoring the formation of paraspeckles.
Vectors pMCSG7-MBP-SFPQ (N-terminal fusion with MBP and His6) and pMCSG7-
SFPQ (untagged SFPQ) were prepared. SFPQ was then expressed using pJ4M-SFPQMBP
(prepared in the lab) and pMCSG7-MBP-SFPQ vectors. The fusion with His6
allowed the protein to be purified by immobilized metal ion affinity chromatography
(IMAC). SFPQ-MBP was already degraded during expressing. MBP-SFPQ was
expressed in sufficient amounts, but degradation occurred during the purification steps.
We used the pMCSG7-SFPQ vector to express untagged SFPQ and isolate it by adding
zinc chloride. Proteolysis occurred upon expression and/or purification. We concluded
that the cause of protein degradation is a large proportion of the disordered structure,
which is more sensitive to proteases. The procedures should therefore be optimized to
reduce the activity of proteases.
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