X-RAY STUDIES ON FAB-FRAGMENT OF TESTOSTERONE SPECIFIC ANTIBODIES

Jarko Valjakka¹, Ari Hemminki², Tuija Teerinen, Kristiina Takkinen² and Juha Rouvinen²

¹ Univ. of Joensuu, Dept. of Chemistry, P.O. Box 111, 80101 Joensuu, Finland.
² VTT Biotechnology and Food Research, P.O. Box 1503, 02044 VTT, Finland.

Keywords: Crystallization, Fab-Fragment, DHEAS, IgG, Testosterone, X-Ray

IgG antibodies are of major importance in the scope of immonoassays. Specific IgG Fab-fragments of high affinity and specificity against hydrophobic testosterone and alike DHEAS molecules have been developed. These steroids are important analyte for many clinical disorders, thus a sensitive assay for it would be very valuable. Protein engineering can be used to modify and improve the properties of antibodies in order to obtain suitable binding characteristics. In addition to the availability of molecular biology tools, information on the protein structure is essential for efficient engineering experiments.

Crystallization and Data collection

Crystals of Fab-fragment of testosterone specific IgG were obtained by vapor diffusion technique at room temperature by using a solution of Peg3350 and Peg8000 with various biological buffers and at wide pH range (3-8). So far, the highest diffractive (2.6A) Fab-fragment crystals contain testosterone and the crystal shape was bipyramid. Native and DHEAS containing crystals diffract only at 3.5A and their crystal shape was sarcophagous. Attempts to solve the three dimensional structure of Fab-fragment by molecular replacement is currently under work. The X-ray diffraction data were measured with a rotating anode generator RU200HB and R-AXIS IIC imaging plate area detector. The data has been processed with DENZO and SCALEPACK programs. The highest crystal diffracted X-rays at 2.6A resolution and belonged to the hexagonal space group P62 (found by the program XPREP) with unit cell constants a=b= 82.8, c=119.29 A. The crystal contains one molecule in the asymmetric unit. The final data set consisted of 14124 unique reflections with Rmerge 7.0% and the data set is 99.9% complete.