Sandbox Reserved 598: Difference between revisions

In determining the three-dimensional structure of the Jak2 protein, three primary methods of analysis were used; protein expression and purification, crystallization, and x-ray data collection. <ref> Lucet, I., Fantino, E., & Styles, M. (2005). The structural basis of janus kinase 2 inhibition by a potent and specific pan-janus kinase inhibitor. Blood, 107, 176-183. doi: 10.1182/blood-2005-06-2413 http://bloodjournal.hematologylibrary.org/content/107/1/176.full.pdf </ref> In the protein expression and purification the Jak2 residue was cloned using pFastBac which uses “two promoters in a single vector for expression of two proteins simultaneously in insect cells”. <ref> http://www.invitrogen.com/1/1/14896-pfastbac-dual.html </ref> The bacmid DNA with the kinase insert was then isolated and put into cells of insect army worms. The cells were then grown, lysed and centrifuged after which the protein was then incubated, separated with gel filtration and fractions were taken for crystallization trials. In these crystallization trials, the protein residue was used to grow crystals via hanging drop vapor-diffusion. The purified protein complex was then mixed with solutions which subsequently formed crystals after one to three days. The crystalized protein was then flash frozen and the structure  determined via molecular replacement and the AmoRe program. <ref> ) Lucet, I., Fantino, E., & Styles, M. (2005). The structural basis of janus kinase 2 inhibition by a potent and specific pan-janus kinase inhibitor. Blood, 107, 176-183. doi: 10.1182/blood-2005-06-2413 http://bloodjournal.hematologylibrary.org/content/107/1/176.full.pdf </ref>