Preliminary Structural Study Of Integrin Activator Talin Protein

Integrin is a very important cell adhesion molecule on the surface of vertebrate cellls,consisting of a,(3 two subunits.integrin inteeacts with the cytoskeletal protein to mediateadhesion between cells and extracellular matrix.integrin is not only a physical link between the extracellular matrix and the intracellular backbone,but also an important compononet of cellular signal transduction.a series of signal events mediated by integrin is one of the important ways for cells to respond the environment.Talin is an important integrin activator that binds and activates integrins.at the same time as a link between the cytoskeleton and transmembrane receptor integrin of the bridge,in the cell adhesion,migration and other process play a key regulatory tole.This research is based on the preliminary study of the laboratory on the basis of the extension and expension.the laboratory study group analyzed the crystal structure of the Talin-F2F3/R9 complex in 2012.this structure shows that in the autoinhibitory state,the integrin binding site F3 on talin binds to its tail fragment R9(1654a.a.-1822a.a.),where the integrin can not be activated.this autoinhibitory complex provides a new perspective for our understanding of Talin's autoinhibitory mechanism,but still dose not reveal the full length sturcture of Talin's autoinhibitory state.As a 270KD protein,Talin can form a dimer through the last a helix of the C-terminus,where the talin protein is autoinhibitory in vivo or two molecules in the two bodies,which are unclear.We proceed from in vitro recombinant expression and natural tissue extraction in order to obtain high quality Talin protein samples.consisting that the Talin protein is difficult to crystallize as a whole,we try to combine the negative staining technique and the three-dimensional reconstruction method of frozen electron microscope to obtain the whole structure of Talin protein in autoinhibitory state.The expression of the protein was undisturbed by negative expression of Talin protein under negative electron microscope.the homogeneity of the protein was improved greatly by the combinated of the biodegradation agent and the density gradient centrifugation method.the quality of the negative electron microscope was higher,and the image contrast was high and the protien particles were obvious.But unfortunately did not get high quality frozen electron microscope pictures.natural tissues extraction,we use ammonium sulfate precipitation method will be a large number of crude agents in some of the target protein precipitation.combined with a variety of separation ability of different anion exchange column,molecular sieve,hydrophobic column,cation exchange column,and finally get a more pure Talin protein samples,negative electron microscopy can see the protein particles...