title: Soluble recombinant CD69 receptors optimized to have an exceptional physical and chemical stability display prolonged circulation and remain intact in the blood of mice.
abstract: We investigated the soluble forms of the earliest activation antigen of human leukocyte CD69. This receptor is expressed at the cell surface as a type II homodimeric membrane protein. However, the elements necessary to prepare the soluble recombinant CD69 suitable for structural studies are a matter of controversy. We describe the physical, biochemical and in vivo characteristics of a highly stable soluble form of CD69 obtained by bacterial expression of an appropriate extracellular segment of this protein. Our construct has been derived from one used for CD69 crystallization by further optimization with regard to protein stability, solubility and easy crystallization under conditions promoting ligand binding. The resulting protein is stable at acidic pH and at temperatures of up to 65 degrees C, as revealed by long-term stability tests and thermal denaturation experiments. Protein NMR and crystallography confirmed the expected protein fold, and revealed additional details of the protein characteristics in solution. The soluble CD69 refolded in a form of noncovalent dimers, as revealed by gel filtration, sedimentation velocity measurements, NMR and dynamic light scattering. The soluble CD69 proved to be remarkably stable in vivo when injected into the bloodstream of experimental mice. More than 70% of the most stable CD69 proteins is preserved intact in the blood 24 h after injection, whereas the less stable CD69 variants are rapidly taken up by the liver.
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.
authors.forename: Ondrej
authors.initials: O
authors.lastname: Vanek
authors.forename: Monika
authors.initials: M
authors.lastname: Nálezková
authors.forename: Daniel
authors.initials: D
authors.lastname: Kavan
authors.forename: Ivana
authors.initials: I
authors.lastname: Borovicková
authors.forename: Petr
authors.initials: P
authors.lastname: Pompach
authors.forename: Petr
authors.initials: P
authors.lastname: Novák
authors.forename: Vinay
authors.initials: V
authors.lastname: Kumar
authors.forename: Luca
authors.initials: L
authors.lastname: Vannucci
authors.forename: Jirí
authors.initials: J
authors.lastname: Hudecek
authors.forename: Katerina
authors.initials: K
authors.lastname: Hofbauerová
authors.forename: Vladimír
authors.initials: V
authors.lastname: Kopecký
authors.forename: Jirí
authors.initials: J
authors.lastname: Brynda
authors.forename: Petr
authors.initials: P
authors.lastname: Kolenko
authors.forename: Jan
authors.initials: J
authors.lastname: Dohnálek
authors.forename: Pavel
authors.initials: P
authors.lastname: Kaderávek
authors.forename: Josef
authors.initials: J
authors.lastname: Chmelík
authors.forename: Lukás
authors.initials: L
authors.lastname: Gorcík
authors.forename: Lukás
authors.initials: L
authors.lastname: Zídek
authors.forename: Vladimír
authors.initials: V
authors.lastname: Sklenár
authors.forename: Karel
authors.initials: K
authors.lastname: Bezouska
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000097:blood* / Q000737:chemistry*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000097:blood* / Q000737:chemistry*; D003460:Crystallization; D019281:Dimerization; D006801:Humans; D037181:Lectins, C-Type; D051379:Mice; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins; D012995:Solubility; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D037181:Lectins, C-Type; D011994:Recombinant Proteins; D000099192:CD69 Antigens