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Quality assessment of recombinant proteins by infrared spectroscopy. Characterisation of a protein aggregation related band of the Ca2+-ATPase
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2014 (English)In: The Analyst, ISSN 0003-2654, E-ISSN 1364-5528, Vol. 139, no 17, 4231-4240 p.Article in journal (Refereed) Published
Abstract [en]

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.
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-107604DOI: 10.1039/c4an00483cISI: 000340703100014OAI: oai:DiVA.org:su-107604DiVA: diva2:750206
Note

AuthorCount:5;

Available from: 2014-09-26 Created: 2014-09-22 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Infrared spectroscopy: a tool for protein characterization
Open this publication in new window or tab >>Infrared spectroscopy: a tool for protein characterization
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Infrared (IR) spectroscopy, which belongs to vibrational spectroscopy, detects the vibrations of molecules, for example, proteins. The absorption of the peptide group gives rise to 9 characteristic bands in the infrared region, named A, B, I-VII, with a decreasing energy or wavenumber (cm-1). Among the 9 bands, amide I, which is mainly caused by C=O stretching vibration, is most sensitive to backbone structure and environment, and therefore can be used for structural analysis. In this thesis, a membrane protein sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) and a self-assembling peptide was studied with IR spectroscopy.  

In the first two papers, IR spectroscopy was used to assess the quality of a recombinant SERCA1a. A yeast-based expression system was applied to express recombinant SERCA1a, and the reaction cycle as well as the structure was analysed with IR spectroscopy. Different reaction intermediates were accumulated under different buffer conditions upon the release of ATP. The results showed that the recombinant protein shared similar IR features compared to the native protein. However, two SERCA1a preparations showed a difference around 1640 cm-1 in the amide I region. Using curve fitting, the band was assigned to β structure, and further investigation indicated that the difference in this region originates from protein aggregation. In the third paper, a co-fitting approach was tested and showed to be a more reliable method for structural analysis, and it can be applied in the biological IR spectroscopy. In the fourth paper, a peptide was computational designed and was predicted to self-assemble to amyloid fibrils, the formation of the fibril was confirmed with both electron microscopy and X-ray diffraction. IR spectroscopy was used to analyze further the structural details and the results support our structural predication. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2016
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-128761 (URN)978-91-7649-407-3 (ISBN)
Public defence
2016-05-27, Magnéli Hall, Arrhenius Laboratory, Svante arrhenius väg 16 B, Stockholm, 10:00 (English)
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Supervisors
Available from: 2016-05-02 Created: 2016-04-04 Last updated: 2017-02-20Bibliographically approved

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