OPTICAL SPECTROSCOPY, MASS SPECTROMETRY AND NMR ARE ESSENTIAL TOOLS IN THE PRODUCTION OF SOLUBLE RECEPTORS OF NATURAL KILLER CELLS

K. Bezouška^1,2, D. Kavan^1,2, O. Vaněk¹, P. Pompach^1,2, L. Mihók^1,2, J. Bílý¹, P. Novák², K. Hofbauerová^2,3, V. Kopecký, Jr.³, M. Nálezková⁴, L. Žídek⁴, V. Sklenář⁴

¹Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, CZ-12840 Praha 2;

²Institute of Microbiology, Academy of Sciences of Czech Repub., Vídeňská 1083, CZ-14220 Praha 4; ³Institute of Physics, Faculty of Mathematics and Physics, Charles University Prague, Ke Karlovu 5, CZ-12116 Praha 2;

⁴National Centre for Biomolecular Research, Faculty of Science, Masaryk University in Brno, Kotlarska 2, 611 37 Brno, Czech Republic.

E-mail: bezouska@biomed.cas.cz

Natural killer (NK) cells represent an important lymphocyte subset critical in the immune response against viruses, certain microorganisms, and tumors. Effector functions of these cells are dependent on the balance between signals transmitted from both activating and inhibitory surface receptors [1]. While the inhibitory receptors interacting with MHC class I as ligand have been well characterized, much less is known about the activating receptors and their ligands [2,3]. In our laboratory we have been producing soluble recombinant forms of the two major activating receptors of NK cells, CD69 and NKR-P1. Our production protocol includes bacterial expression of the extracellular portion of the above receptors. Precipitation into the inclusion bodies is followed by in vitro refolding and purification by a combination of ion exchange, hydrophobic, and gel permeation chromatographies. The purified proteins appeared to be homogeneous on SDS polyacrylamide gel electrophoresis displaying a notable shift towards higher mobility under the nonreducing conditions. They are refolded in the form of noncovalent dimers as revealed by gel filtration and cross-linking experiments. The verification of the proper folding after in vitro procedure is critical for all the subsequent protein applications. For the initial assessment of the refolding efficiency, we use a combination of the UV, FT-IR and Raman spectroscopy. UV spectroscopy serves as a good first test of the stability of the produced protein (thermal denaturation curves measured in varying chemical environments). Vibrational spectroscopy, on the other hand, is able to estimate the content of secondary structure elements (such as a-helices, b-sheets etc.) as well as structural details of certain amino acids such as cysteines. For the final verification of the folding status, high resolution methods that look onto proteins in the high magnetic fields are necessary. We use ion Fourier transform-ion cyclotron resonance mass spectrometry to look at the identity of the entire protein and the number of closed disulfide bonds. Measurement of the 1H – 15N HSQC spectra in a 600 MHz NMR spectrometer using the uniformly ¹⁵N labeled protein provides the definitive evidence for the proper folding and long-term stability of the protein preparations. The above measurements revealed that our preparations of soluble rat and human CD69 protein is stable for weeks / months at temperatures between 4 °C and 30 °C, while the short term stability up to 80 °C is worth mentioning [4]. Also, our most recent measurements of 1H – 15N HSQC spectra of the rat NKR-P1A protein points to the success of the currently employed refolding protocol. The use of NMR as well as protein crystallography and other methods for the ligand identification and verification experiments is under progress.

1. R.K. Strong, Mol. Immunol. 38, (2002), 1029.

2. K. Bezouška, J.Biotechnol. 90, (2002), 269.

3. K. Bezouška, Collect.Czech.Chem.Commun. 69, (2004), 535.

4. J. Pavlíček., D. Kavan, P. Pompach, K. Bezouška, Biochem. Soc. Trans. 32, (2004), 1124.

This research has been supported by Ministry of Education of Czech Republic (MSM 0021620808), by Institutional Research Concept No. AVOZ 50200510 for the Institute of Microbiology,and by Grant Agency of the Academy of Sciences of the Czech Republic No. A5020403.