[Improved the solubility of maize uroporphyrinogen III methyltransferase as the red fluorescent indicator by site-directed mutagenesis].
S-adenosylmethionine-dependent uroporphyrinogen III methyltransferase (SUMT) is a novel red fluorescence indicator. However, the production of SUMT in Escherichia coli is restricted by its relatively low solubility, and little is known about the red fluorescent materials that are associate with SUMT. Two individual SUMT mutations, L166A and L88R/L89G double mutant were produced by site-directed mutagenesis. Both mutants were overexpressed in E. coli and purified by Ni-NTA chromatography. The reddish mixtures isolated from the purified L88R/L89G double mutant were analyzed by UV-visible spectra scanning and mass analysis(MS). The L88R/L89G double mutant has enzymatic activity in vivo, whereas L166A mutant loses the activity. Trimethylpyrrocorphin is identified as the main constituent in the isolated pigments. The purified L88R/L89G mutant increases protein solubility, which is applied potentially as the fluorescent indicator denoting the solubility of protein fusion partner.
Pan
Hai-Yun
HY
School of Life Sciences, Anhui Agricultural University, Hefei , 230036, China.
Cheng
Ying
Y
Zhu
Su-Wen
SW
Fan
Jun
J
D019943:Amino Acid Substitution; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D013058:Mass Spectrometry; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D010860:Pigments, Biological / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D010860:Pigments, Biological; D010940:Plant Proteins; D011994:Recombinant Proteins; D008780:Methyltransferases; C060508:uroporphyrin-III C-methyltransferase