A. TGF-beta related proteins. We have crystallized a TGF-beta1 complex with a neutralizing antibody 1D11.16 in order to delineate the surface loops of TGF-beta1 that mediate the receptor binding. The best crystals are grown in the presence of 8 - 10% PEG 8000, 20% glycerol, 0.2 M sodium acetate and 0.1 M sodium citrate at pH 5.6. A typical crystal measures 0.1 x0.1 x0.3 mm in size and it diffracts to about 2.9 angstroms resolution. Using SDS gel electrophoresis, we have shown the presence of both TGF-beta1 and 1D11.16 in this crystal form. We have also crystallized a complex of TGF-beta and its type II receptor. The existance of the complex has been demonstrated by SDS gel electrophoresis. Currently, the size of these crystals are still small and further improvement in crystallization condition is needed to obtain data collection quality crystals. B. Immunoglobulin Fc receptors. A method has been developed in this laboratory to over express soluble Fc receptors of human FcgammaRII, FcgammaRIII and a mouse FcgammaRII. Cells are cultured on the surface of cytodex 2 beads as microcarriers. A three step scale-up protocal is implemented in order to obtain milligram quantity of proteins. C. Molybdopterin containing formate dehydrogenase H from E. coli. Due to the extreme oxygen sensitivity of FDH, the enzyme was purified and crystallized under anaerobic conditions. Crystals suitable for X-ray diffraction studies wer e obtained in a space group P4/12/12 with cell dimensions a=b=146.2 angstroms, c=83.2 angstroms. We have developed a cryofreezing protocol for the FDH data collection to avoid oxidation of the enzyme. The native and a number of heavy atom derivative screening data sets have been collected using our lab source x-ray facilities. Among the potential heavy atom derivatives, we solved a major binding site of K2PtCl/4 by inspecting its difference Patterson map and other derivatives by cross Fourier method. A set of multiple wavelength diffraction data were collected using Brookhaven synchrotron facility for phase determinations. D. Blood-type converting enzymes. We have crystallized two blood-type converting enzymes, an alpha-galactosidase (alpha-Gal) which converts A- to O-type blood and an alpha-N-acetylgalactosaminidase (alpha-NAGal) that converts B- to O-typeblood. Crystals of `-NAGal grow to a size of 0.85mm x 0.28mm x 0.22mm from a solution containing 1.5M ammonium sulfate or lithium sulfate and 0.1 M sodium acetate at pH 5.6. They diffract to 2.8 angstroms and belong to the spae group P4/12/12 with unit cell dimensions of a=b-72.1 angstroms, c=176.3 angstroms (alpha=beta=gamma=90 degrees). Native and a number of heavy atom derivative data sets have been collected under cyrofreezing conditions both in our lab and in NSLS for MIR and anomalous phase determination.
Showing the most recent 10 out of 22 publications
