[ccp4bb] the lysozyme of membrane proteins?
Hello, I am developing an undergraduate biochemistry lab class and would like to incorporate experiments with membrane proteins. Does anyone have suggestions on membrane proteins that are relatively easy to express, purify, and assay? Bonus points for crystallizable! At the moment, my leading candidate is aquaporin AqpZ from E. coli. I am planning to express the membrane protein as a GFP fusion so students can easily follow it through the course of the labs. Thank you, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu
Re: [ccp4bb] the lysozyme of membrane proteins?
Wouldn't bacteriorhodopsin be a good choice, especially since it's colored and easy to express? Seems pretty expensive for a class, though, with all the detergents involved... JPK On Tue, Sep 11, 2012 at 4:18 PM, Ho Leung Ng h...@hawaii.edu wrote: Hello, I am developing an undergraduate biochemistry lab class and would like to incorporate experiments with membrane proteins. Does anyone have suggestions on membrane proteins that are relatively easy to express, purify, and assay? Bonus points for crystallizable! At the moment, my leading candidate is aquaporin AqpZ from E. coli. I am planning to express the membrane protein as a GFP fusion so students can easily follow it through the course of the labs. Thank you, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] the lysozyme of membrane proteins?
Two additional suggestions: 1. Photosynthetic Reaction Center from *R. sphaeroides* might be up your alley. Similar to bacteriorhodopsin, no need for a GFP tag on this one as it's colored. See the following publications: Wallace et al. Monoolein Lipid phases as incorporation and inrichment materials for membrane protein crystallization PLoS One. Aug 31 2011. Kors et al. Effects of impurities on membrane-protein crystalllzation in different systems Acta Cryst D Biol Crystallogr. Oct 2009. p1062-73. 2. The beta barrel transmembrane domain of intimin would work as well. Expresses in standard BL21 DE3 *E. coli* cells, 3 step purification, crystallizes readily in LCP, and is stable for months at a time at 4 degrees (not colored though): Fairman et al. Crystal Structures of the outer membrane domain of intimin and invasin from enterohemorrhagic *E. coli* and enteropathogenic *Y. pseudotuberculosis*. Structure. Jul 3 2012. p 1233-43. Cheers, Jim On Tue, Sep 11, 2012 at 2:18 PM, Ho Leung Ng h...@hawaii.edu wrote: Hello, I am developing an undergraduate biochemistry lab class and would like to incorporate experiments with membrane proteins. Does anyone have suggestions on membrane proteins that are relatively easy to express, purify, and assay? Bonus points for crystallizable! At the moment, my leading candidate is aquaporin AqpZ from E. coli. I am planning to express the membrane protein as a GFP fusion so students can easily follow it through the course of the labs. Thank you, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructures http://www.emeraldbiostructures.com/ Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.com jfair...@embios.com
Re: [ccp4bb] the lysozyme of membrane proteins?
Hi, Just for info if you were to use the LCP method (a course by itself), check this about OmpF (membrane lysozyme): http://www.sciencedirect.com/science/article/pii/S1047847712000834 bR protein sometimes takes weeks to give crystals and people prefer the dark (depends on the conf state).. but good idea for spectro assays (check reaction centres/light-harvesting complexes too). Beta barrels are very stable too. If you want to use the GFP fusion and you want to cleave it, it might add extra time and steps to the students than going directly with a GFP free his-tagged protein to trials. Regards Toufic El Arnaout Membrane Structural and Functional Biology Group Trinity College Dublin On Tue, Sep 11, 2012 at 10:18 PM, Ho Leung Ng h...@hawaii.edu wrote: Hello, I am developing an undergraduate biochemistry lab class and would like to incorporate experiments with membrane proteins. Does anyone have suggestions on membrane proteins that are relatively easy to express, purify, and assay? Bonus points for crystallizable! At the moment, my leading candidate is aquaporin AqpZ from E. coli. I am planning to express the membrane protein as a GFP fusion so students can easily follow it through the course of the labs. Thank you, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu
Re: [ccp4bb] the lysozyme of membrane proteins?
The multidrug efflux transporter AcrB could be suitable including crystallization (both recombinant with His-tag and endogenous). Extracts, solubilizes, purifies and crystallizes in DDM, so it is fairly inexpensive to repeat in bulk compared to other detergents. Assays might be a bit complicated. And it is impressive in architecture too, forming a trimer with 12 TM in each monomer, with 70A span in the periplasm and 50A in the inner membrane and very little in the cytoplasm. Thanks, Debanu. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ho Leung Ng Sent: Tuesday, September 11, 2012 2:19 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] the lysozyme of membrane proteins? Hello, I am developing an undergraduate biochemistry lab class and would like to incorporate experiments with membrane proteins. Does anyone have suggestions on membrane proteins that are relatively easy to express, purify, and assay? Bonus points for crystallizable! At the moment, my leading candidate is aquaporin AqpZ from E. coli. I am planning to express the membrane protein as a GFP fusion so students can easily follow it through the course of the labs. Thank you, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu
