Advanced Methods in Structural Biology by Toshiya Senda, Katsumi Maenaka

By Toshiya Senda, Katsumi Maenaka

This ebook presents functional details on an entire set of protein experiments for complicated structural biology, reminiscent of X-ray crystallography, NMR, electron microscopy, complex mass spectroscopy, and floor plasmon resonance, in addition to a wide selection of expression platforms together with eukaryotic and in vitro expression. some time past decade, structural genomics reports have driven ahead the improvement of automatic tools within the box of structural biology, in spite of the fact that there's an expanding want for the structural research of adverse goals, reminiscent of huge protein complexes and membrane proteins, that are difficult to accomplish utilizing traditional computerized tools, and require wisdom that is going past general protein chemistry protocols. to handle those difficulties and to aid researchers enhance novel tools, this quantity presents examples of the advance of latest protein research equipment and their theoretical historical past. This booklet rather appeals to graduate scholars, postdoctoral researchers, younger investigators wishing to realize a greater knowing of the speculation in the back of experiments, and people looking additional complex, sensible structural biology methods.

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Chemicals: isopropyl β-D-thiogalactopyranoside (IPTG), X-gal. 2 Expression in Insect Cells Insect cell lines: Sf9, Sf21, High Five. Media: Sf900 II SFM (Invitrogen), Fetal Bovine Serum (HyClone), Express Five SFM. Transfection reagents: X-tremeGENE HP Transfection Reagent (Roche). Flasks: Erlenmeyer flasks. Safety cabinet: biological safety cabinet (Sanyo). Shaker incubator: Bioshaker (Taitec). 3 Expression in Silkworm Silkworms: Bombyx mori (Kinshu  Showa race) (Ehime-Sanshu). Feed: synthetic diet (Ehime-Sanshu).

Details of this system are described in Sect. 4. 2 Cell Lines and Virus Types Sf9, Mimic Sf9, Sf21, SF+, and High Five cells are basic cell lines used for recombinant protein production. Sf9, Mimic Sf9, Sf21, and SF+ cells are derived from Spodoptera frugiperda. Sf9 cells are the standard cell lines, but Sf21 cells usually show higher protein production than do Sf9 cells. SF+ cells are suitable for suspension cultures and are thus used for large-scale protein production. Mimic Sf9 cells are a derivative of Sf9 cells that stably express mammalian glycosyltransferases.

Reeves PJ, Callewaert N, Contreras R, Khorana HG (2002) Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible Nacetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc Natl Acad Sci U S A 99:13419–13424. 212519299 18. Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23:567–575. 1038/ nbt1095 19.

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