ACCURACY AND INFORMATIONAL CONTENT OF SPECTROSCOPIC METHODS FOR DETERMINATION OF THE SECONDARY STRUCTURE OF PROTEINS EXPRESSED AS A PART OF THE GENE FUSION SYSTEM
V. Kopecký Jr.1,2, K. Hofbauerová2,3 and V. Baumruk1
1 Institute of Physics, Charles University, Ke Karlovu 5, Prague 2, CZ-12116, Czech Republic, firstname.lastname@example.org
2 Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, Prague 2, CZ-12840, Czech Republic
3 Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 4, CZ-14220, Czech Republic
The main aim of this study is to characterize accuracy of the standard spectroscopic methods, i.e. circular dichroism (CD), Fourier transform infrared (FTIR) and Raman spectroscopy, for determination of the secondary structure of the proteins, which are co-expressed with a soluble protein. In many cases when proteins are expressed in gene fusion systems it seems useful to keep a soluble protein covalently bound to the studied protein. The reason is that an acquisition of the desired protein represents thrombin cleavage from the fusion protein, which cause smaller yield of the purified protein. Moreover, the cleavage site could exist in the expressed protein or the protein alone is insoluble.
We examined the glutathione S-transferase (GST) gene fusion system, which belongs to the most widely used systems for expression, purification and detection of proteins in E. coli. The system is based on intracellular expression of genes or gene fragments co-expressed with S. japonicum GST . The model protein that was co-expressed with GST in our study was N-domain of H4–H5 loop of Na+/K+-ATPase. The mass ratio of the N-domain to GST protein was approximately 1:1. Prepared samples were N-domain alone, GST alone and GST covalently bond to N-domain. It gives a chance to compare results from spectroscopic measurements of the independent proteins, which should be the sum for the GST covalently bond to N-domain.
CD spectra were analysed for the secondary structure content by means of the Dichroweb website . All published analyses of the Raman amide I and amide III bands and most of methods for FTIR amide I and II bands were used under Matlab® as a part of our Vibrational Spectroscopy Toolbox & Application . Dependence of informational content and accuracy of the Raman spectra on a protein concentration has been also examined.
Support from the Ministry of Education of the Czech Republic (No. MSM113100001, No. MSM113200002) and from Grant Agency of the Czech Republic (grants Nos. 204/01/0254, 204/01/1001, 309/02/1479) is gratefully acknowledged.
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