Crystallization and diffraction analysis of the serpin IRS-2 from the hard tick Ixodes ricinus.
IRS-2 from the hard tick Ixodes ricinus belongs to the serpin family of protease inhibitors. It is produced in the salivary glands of the tick and its anti-inflammatory activity suggests that it plays a role in parasite-host interaction. Recombinant IRS-2 prepared by heterologous expression in a bacterial system was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the primitive tetragonal space group P4(3) and diffracted to 1.8 Å resolution. Mass-spectrometric and electrophoretic analyses revealed that IRS-2 was cleaved by contaminating proteases during crystallization. This processing of IRS-2 mimicked the specific cleavage of the serpin by its target protease and resulted in a more stable form (the so-called relaxed conformation), which produced well diffracting crystals. Activity profiling with specific substrates and inhibitors demonstrated traces of serine and cysteine proteases in the protein stock solution.
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám. 2, 16610 Praha 6, Czech Republic.
Zuzana
Z
Kovářová
Jindřich
J
Chmelař
Miloslav
M
Sanda
Jiří
J
Brynda
Michael
M
Mareš
Pavlína
P
Rezáčová
D000818:Animals; D003460:Crystallization; D018360:Crystallography, X-Ray; D018884:Ixodes / Q000737:chemistry*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D015843:Serpins / Q000737:chemistry*
D015843:Serpins