Dear All:
Need an explanation...I am working with maize GSTs
(GSTI, III and Bronze2) and Petunia GST namely
An9. I have also created point mutations in these
particular genes, and am in the process of doing
in vitro CDNB activity assays with these
recombinant proteins (cloned in pQE30 vector as 6X
his tagged proteins and are expressed in either
M15(pRep4) or JM109 strains).
The funny thing I am observing since few
extractions (Extraction Buffer: Na-phosphate +20%
glycerol, pH 6.8) is that I get a "pink bacterial
extract"!!! Now An9 is involved in anthocyanin
(pink pigment) sequestration in petunia and so
does the maize GSTIII and Bz2..In addition, the
pink coloration is observed exclusively in
recombinant unmutated and mutated An9 and mutant
GSTIII extracts. I am unable to comprehend why the
pink coloration of the bacterial extract!!! Like
in plants, bacteria do not synthesize
anthocyanin!!!
Could anyone think of a plausible explanation...
Also my observation, the pink An9 (unmutated)
extracts have reduced CDNB activity!!..
Thanks a ton..
Savita
