Expression, purification and crystallization of native and selenomethionine labeled Mycobacterium tuberculosis FGD1 (Rv0407) using a Mycobacterium smegmatis expression system.
FGD1 is an F(420)-dependent glucose-6-phosphate dehydrogenase from Mycobacterium tuberculosis that has been shown to be essential for activation of the anti-TB compound PA-824. Initial attempts to produce recombinant FGD1 using Escherichia coli as a host was unsuccessful, but when the alternative host Mycobacterium smegmatis was used, soluble protein yields of 7 mg/L of culture were achieved. Both native and selenomethionine-substituted FGD1 were obtained by culturing M. smegmatis in autoinduction media protocols originally developed for E. coli. Using these media afforded the advantages of decreased handling, as cultures did not require monitoring of optical density and induction, and reduced cost by removing the need for expensive ADC enrichment normally used in mycobacterial cultures. Selenomethionine was efficiently incorporated at levels required for multiwavelength anomalous diffraction experiments used in crystal structure determination. As far as we are aware this is the first protocol for preparation of selenomethionine-substituted protein in mycobacteria. Native and selenomethionine-labeled FGD1 were successfully crystallized by vapor diffusion, with the crystals diffracting to 2.1 Angstrom resolution.
Bashiri
Ghader
G
Maurice Wilkins Center for Molecular Biodiscovery, Laboratory of Structural Biology, School of Biological Sciences, University of Auckland, Private Bag 92019, New Zealand.
Squire
Christopher J
CJ
Baker
Edward N
EN
Moreland
Nicole J
NJ
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D018929:Cell Culture Techniques; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D005954:Glucosephosphate Dehydrogenase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D020102:Mycobacterium smegmatis / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012645:Selenomethionine / Q000032:analysis* / Q000378:metabolism
D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins; D012645:Selenomethionine; D005954:Glucosephosphate Dehydrogenase