Expression, Crystallization And Crystallization Of Cell Adhesion Molecule Nectin-4and Nectin-2

Nectins are cell adhesion molecules (CAMs), which belong to the immunoglobulin superfamily and play essential roles in Ca2+-independent intracellular interactions. The nectin family has four members:nectin-1,-2,-3and-4. Nectin-4could support measles virus entry, and the lateral spread of the virus in well-differentiated primary human airway epithelial sheets. Human nectin-4has a high expression in cancer cells, such as lung cancer, breast cancer and ovarian cancer. Of note, the high expression of nectin-4in cancer cells and its strong specificity for measles virus also remind us of a potential function of oncolysis for nectin-4. Nectin-2can also be used as receptors for virus entry, the structure of nectin-2V domain clearly displays a homodimer form. The interaction of nectin-2V domain may depend on the homodimer. To verify this hypothesis, we expressed the mutant protein of nectin-2V monomer for crystallization. We expressed proteins by Escherichia coli expression system. The proteins were crystallized using both the sitting-drop and hanging-drop vapor-diffusion method. Our research could provide a theoretical basis for the structural elucidation of nectin-4V and nectin-2V monomer (FAMP).The major findings were as dollows:(1) Molecular cloning of recombinant plasmid of nectin-4V-pET21a(+).(2) Expression of nectin-4V domain(residues32to145) by Escherichia coli expression system as a soluble protein with His-tag. The protein was then purified with Ni-NTA and molecular sieve chromatography.(3) Crystallization by using the sitting-drop vapor-diffusion method at18℃in a condition consisting of5%v/v Tacsimate,0.1M HEPES pH7.0,10%w/v Polyethylene glycol monomethyl ether5,000. The nectin-4V crystals diffracted to1.8A resolution and belonged to space group P21, with unit-cell parameters a=33.1A, b=51.7A, c=56.9A, β=94.7°.(4) Nectin-2V FAMP was expressed in E.coli as inclusion bodies, which were subsequently refolded and purified with molecular sieve chromatography.(5) Crystallization by using the hanging-drop vapor-diffusion method at18℃in a condition consisting of0.1M Tris hydrochloride PH8.5,2.0M Ammonium Sulfate. The nectin-2V FAMP crystals diffracted to1.8A resolution and belonged to space group P212121, with unit-cell parameters a=43.3A, b=45.8A, c=52.6A, p=90.0°.