|Cloning, Expression, and Crystallization of Adenylate Kinase for Dynamic Studies using the X-ray Free Electron Laser|
|Presenter||Jose Luis Olmos, Jr.|
|Full Author List||Enrique Martinez, III, Jonathan A. Clinger, George N. Phillips, Jr.|
Adenylate kinase is a small, phosphotransferase enzyme of an approximate molecular weight of 24kDa, depending on the source organism, that catalyzes the reaction 2 ADP ßà ATP + AMP, interconverting adenine nucleotides. Adenylate kinase is ubiquitous in all living organisms, given its important reaction involving energy conversion for cellular processes. This project involves the production of adenylate kinase from three different source organisms, Bacillus subtilis, globisporus, and stearothermophilus. Adenylate kinases from these three different strains are highly conserved with 66% - 74% amino acid identity, however, their optimal activity correlates to the environmental temperatures in which the host species is found, in that globisporus, psychrophile, subtilis, mesophile, and stearothermophilus, thermophile, thrive at low, moderate, and high temperatures, respectively. Having high structural similarities, with varying amino acid composition, but differing activity temperature profiles make these adenylate kinases great models for dynamic studies. The long-term interest of this study is in growing nanocrystals of these three different kinases and using the XFEL (X-ray free electron laser) to elucidate the motions of the molecular machinery of these enzymes at an atomic level of detail, by capturing many snapshots of the nanocrystals at non-cryogenic temperatures, including 60°C where the thermophile normally lives, as they are destroyed by the XFEL.
The adenylate kinase genes were synthesized by IDT. They were then PIPE cloned into vector pNIC28-Bsa4, which includes a 6X-His tag for purification, and transformed into B834(DE3) competent E.coli cells. The cells were grown in defined, auto-inducing media, which induced the over-expression of the target protein. The cells were then harvested by centrifugation and lysed by sonication. After an initial IMAC or immobilized metal ion affinity chromatography column purification, using Ni-NTA resin, one liter cultures of each adenylate kinase produced protein yields of about 35mg for subtilis, 34mg for globisporus and 14mg for the stearothermophilus. The 6X-His tag was cleaved using TEV protease, or Tobacco Etch Virus protease, and a subtractive IMAC was performed to remove the TEV protease and cleaved 6X His-tag. The final purification step was size exclusion chromatography using G200 Sephadex gel filtration. Crystal trays for various conditions of each of the adenylate kinases using the Mosquito LCP were set up and X-ray diffraction data will be collected.
|Funding Acknowledgement||This work was supported by NSF 1231306, BioXFEL.|