Human recombinant laminin-binding protein: Isolation, purification, and crystallization

The mRNA of the precursor of laminin-binding protein (LBP) was isolated fro m a human embryo kidney cell line and cloned. The determined sequence of th e LBP gene showed complete identity with the LBP genes isolated from human lung and large intestine cells. The human LBP was expressed by E. coli cell s, and it was purified using Ni-NTA-Sepharose chromatography. The mobility of the homogeneous recombinant human laminin-binding protein on SDS-PAGE wa s 43 kD. A mixture of eight murine monoclonal antibodies, the MPLR Pool aga inst LBP, reacted with the recombinant LBP in Western blot. The interaction of the antiidiotypical antibodies 10H10 and E6B provided evidence that the epitope binding to protein E of the tick-borne encephalitis (TBE) virus is also preserved on the human recombinant LBP. Enzyme immunoassay confirmed the ability of the recombinant LBP to interact with protein E of TBE virus. The biological activity of the recombinant LBP allowed us to perform X-ray analysis of the spatial arrangement of the LBP molecule using the recombin ant protein. For this purpose, crystals of the human LBP were obtained by t he standing drop version of the pore diffusion technique. The crystals appr opriate for X-ray structural analysis were 0.3 x 0.1 x 0.05 mm in size. The X-ray diffraction field of the crystal extended to 2.5 Angstrom.