AN EASY OPTIMALIZATION METHOD FOR IN VITRO PROTEIN REFOLDING – THE CASE OF CD69

 

O. Vaněk1, J. Pavlíček1, V. Kopecký Jr.3, K. Bezouška1,2

 

1 Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8,

 CZ-12840 Prague 2.

2 Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 2.

3 Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 5, CZ-12116 Prague 2.

 

In protein biochemistry we must often solve a problem of renaturation (refolding) of proteins of our interest. Especially in the case of bacterial recombinant proteins, which are often obtained in insoluble form (the inclusion bodies), we must find an optimal way to transfer the protein molecules from denaturating agents to native conditions. We use an easy optimalization method for in vitro refolding of such proteins, which is critical and demanding task. Successful renaturation of the protein is a multiparameter optimalization procedure that involves finding of certain discrete conditions, such as presence of suitable additives (i.e. arginin), pH, ionic strength and redox potential of the system. These parameters are optimalized in semimicroquantitative way. Here we present results for CD69 protein [1], an early activation antigen of human lymphocytes. Efficiency of the refolding was analyzed by gel filtration and SDS-PAGE. Secondary structure of receptor CD69 prepared via such optimalized protocol was determined by drop coating deposition Raman spectroscopy method [2].

 

This work was supported by Ministry of Education of the Czech Republic No. MSM 0021620808, by Institutional Research Concept No. AVOZ 50200510, by Grant Agency of the Czech Republic No. 301/05/P567, and by Grant Agency of the Academy of Sciences of the Czech Republic No. A5020403.

 

[1] J. Pavlíček, B. Sopko, R. Ettrich, V. Kopecký Jr., V. Baumruk, P. Man, V. Havlíček, M. Vrbacký, L. Martínková, V. Křen, M. Pospíšil, K. Bezouška, Biochemistry 42 (2003) 9295-9306.

[2] D. Zhang, Y. Xie, M. F. Mrozek, C. Ortiz,V. J. Davisson, D. Ben-Amotz, Anal. Chem. 75 (2003) 5703–5709.