蛋白质晶体学-2010精编.ppt

* * * * * * * * * * * Circular Dichroism (CD) Spectroscopy Circular Dichroism (CD) is observed when optically active matter absorbs left and right hand circular polarized light slightly differently. It is measured with a CD spectropolarimeter, which is relatively expensive (~$70k). The instrument needs to be able to measure accurately in the far UV at wavelengths down to 190-170 nm. In addition, the difference in left and right handed absorbance A(l)- A(r) is very small (usually in the range of 0.0001) corresponding to an ellipticity of a few 1/100th of a degree. The CD is a function of wavelength. CD spectra for distinct types of secondary structure present in peptides, proteins and nucleic acids are different. The analysis of CD spectra can therefore yield valuable information about secondary structure of biological macromolecules. See reference [1] for a review. * To study the crystal structure of a protein or protein complex, enough highly purified protein to crystallize is the vital step. Since different vectors, different tags, different bacteria strains or different eukaryotic host cells may influence the expression and behavior of the recombinant protein, to obtain a recombinant protein, sometimes I have to try different ways one by one. No try, no confirm. The following 5 slides show some different vectors used for expressing my target proteins in varies host cells. * This is one of easiest yeast expression system vector. The cloned gene will recombinant to yeast chromosome and the expression is induced by methanol. * This is a easy-handling vector for insect cell expression. The advantage of this vector system is: before the virus infects the cells, it has been confirmed recombinant, so the recombinant efficiency would be 100% theoretically in insect cell. * The human 293 cell line is widely used for r-protein production as it offers many advantages such as high transfection yields with most gene transfer vehicles, is easily grown in suspension cult