Quality assessment of recombinant proteins by infrared spectroscopy. Characterisation of a protein aggregation related band of the Ca2+-ATPase
2014 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 139, no 17, 4231-4240 p.Article in journal (Refereed) Published
Infrared spectroscopy was used to characterise recombinant sarcoplasmic reticulum Ca2+-ATPase (SERCA1a). In the amide I region, its spectrum differed from that of Ca2+-ATPase prepared from rabbit fast twitch muscle below 1650 cm(-1). A band at 1642 cm(-1) is reduced in the spectrum of the recombinant protein and a band at 1631 cm(-1) is more prominent. By comparison of amide 1 band areas with the known secondary structure content of the protein, we assigned the 1642 cm(-1) band to beta-sheet structure. Further investigation revealed that the 1642 cm(-1) band decreased and the 1631 cm-1 band increased upon storage at room temperature and upon repeated washing of a protein film with water. Also protein aggregates obtained after solubilisation of the rabbit muscle enzyme showed a prominent band at 1631 cm(-1), whereas the spectrum of solubilised ATPase resembled that of the membrane bound protein. The spectral position of the 1631 cm(-1) band is similar to that of a band observed for inclusion bodies of other proteins. The findings show that the absence of the 1642 cm(-1) band and the presence of a prominent band at 1631 cm(-1) indicate protein aggregation and can be used as a quality marker for the optimisation of recombinant protein production. We conclude that recombinant production of SERCA1a, storage at room temperature, repeated washing and aggregation after solubilisation all modify existing beta-sheets in the cytosolic domains so that they become similar to those found in inclusion bodies of other proteins.
Place, publisher, year, edition, pages
2014. Vol. 139, no 17, 4231-4240 p.
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:su:diva-107604DOI: 10.1039/c4an00483cISI: 000340703100014OAI: oai:DiVA.org:su-107604DiVA: diva2:750206