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Issue 17, 2014
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Quality assessment of recombinant proteins by infrared spectroscopy. Characterisation of a protein aggregation related band of the Ca2+-ATPase

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Abstract

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 I band areas with the known secondary structure content of the protein, we assigned the 1642 cm−1 band to β-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 β-sheets in the cytosolic domains so that they become similar to those found in inclusion bodies of other proteins.

Graphical abstract: Quality assessment of recombinant proteins by infrared spectroscopy. Characterisation of a protein aggregation related band of the Ca2+-ATPase

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Article information


Submitted
14 Mar 2014
Accepted
22 May 2014
First published
22 May 2014

Analyst, 2014,139, 4231-4240
Article type
Paper
Author version available

Quality assessment of recombinant proteins by infrared spectroscopy. Characterisation of a protein aggregation related band of the Ca2+-ATPase

C. Li, S. Kumar, C. Montigny, M. le Maire and A. Barth, Analyst, 2014, 139, 4231
DOI: 10.1039/C4AN00483C

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