title: Quality assessment of recombinant proteins by infrared spectroscopy. Characterisation of a protein aggregation related band of the Ca²⁺-ATPase.
abstract: Infrared spectroscopy was used to characterise recombinant sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a). In the amide I region, its spectrum differed from that of Ca(2+)-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.
authors: Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, SE-106 91 Stockholm, Sweden. Andreas.Barth@dbb.su.se.
authors: Chenge
authors: C
authors: Li
authors: Saroj
authors: S
authors: Kumar
authors: Cédric
authors: C
authors: Montigny
authors: Marc
authors: M
authors: le Maire
authors: Andreas
authors: A
authors: Barth
mesh_terms: D000818:Animals; D066329:Protein Aggregates; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D014867:Water / Q000737:chemistry
chemical_list: D066329:Protein Aggregates; D011994:Recombinant Proteins; D014867:Water; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases