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. Author manuscript; available in PMC: 2010 Jan 1.
Published in final edited form as: Microbiol Res. 2008 Oct 23;164(1):1–8. doi: 10.1016/j.micres.2008.08.006

Figure 4. Interaction of YgfD and Sbm in vitro.

Figure 4

A. Expression and purification of His-tagged Sbm (left panel) and YgfD (right panel) run on 12% SDS-PAGE and stained by Coomassie blue. For both: lane 1, total cell lysate; lane 2, final wash fraction before elution; lane 3, eluate containing purified protein. B. Native 4–20% polyacrylamide gel containing Sbm apoenzyme incubated with increasing molar equivalents of YgfD pre-incubated with GMPPNP. Sbm ran at ~80 kDa monomer and ~160 kDa dimer (Sbm only lane), while YgfD (~40 kDa) was run off this gel but not seen previously (YgfD only lane). Note appearance of species (Complex 1 and Complex 2) with masses in excess of the 232 kDa MW standard at increasing YgfD ratio (maximal band shift obtained at 4:1 YgfD:Sbm incubation mix). C. Purified Sbm, YgfD, or an equal mixture of the two, were analyzed by gel filtration. Proteins were run in the presence of MW standards (bovine milk α-lactalbumin (14.2 kDa), bovine erythrocytes carbonic anhydrase (29 kDa), chick egg albumin (45 kDa), BSA (66 kDa – monomer, 132 kDa – dimer), and Jack Bean urease (272 kDa – trimer, 545 kDa – hexamer)) to prevent precipitation and loss of proteins during sieving. Panel 1, MW standards with molecular masses in kDa indicated above each peak; Panel 2, Sbm plus MW standards, with Sbm monomer (“α”) and dimer (“αα”) peaks filled in black; Panel 3, YgfD plus MW standards with YgfD peak in black; Panel 4, YgfD plus Sbm with each protein and putative complex (“ααyy”) filled in black. Peaks in black were determined by comparison with peaks in Panel 1. The x- and y-axis refer to time (min) and elution profile at OD280, respectively, for all charts. D. Native 4–20% polyacrylamide gel containing different combinations of YgfD and Sbm with or without prior incubation to cofactor/coenzyme demonstrating that only YgfD pre-incubated with GMPPNP results in complex formation with Sbm. Lane 1, YgfD:GMPPNP & Sbm:AdoCbl; Lane 2, YgfD:GMPPNP & Sbm:HOCbl; Lane 3, YgfD:GMPPNP & Sbm; Lane 4, YgfD & Sbm:AdoCbl; Lane 5, YgfD & Sbm:HOCbl; Lane 6, YgfD & Sbm; Lane 7, Sbm:AdoCbl; Lane 8, Sbm:HOCbl; Lane 9, Sbm; Lane 10, YgfD:GMPPNP; Lane 11, YgfD; Lane 12, YgfD:GTP; Lane 13, YgfD:GDP; Lane 14, YgfD:GTP & Sbm; Lane 15, YgfD:GDP & Sbm.