Assay and heterologous expression in Pichia pastoris of plant cell wall type-II membrane anchored glycosyltransferases.
Two Arabidopsis xylosyltransferases, designated RGXT1 and RGXT2, were recently expressed in Baculovirus transfected insect cells and by use of the free sugar assay shown to catalyse transfer of D-xylose from UDP-alpha-D-xylose to L-fucose and derivatives hereof. We have now examined expression of RGXT1 and RGXT2 in Pichia pastoris and compared the two expression systems. Pichia transformants, expressing soluble, secreted forms of RGXT1 and RGXT2 with an N- or C-terminal Flag-tag, accumulated recombinant, hyper-glycosylated proteins at levels between 6 and 16 mg protein * L(-1) in the media fractions. When incubated with 0.5 M L-fucose and UDP-D-xylose all four RGXT1 and RGXT2 variants catalyzed transfer of D-xylose onto L-fucose with estimated turnover numbers between 0.15 and 0.3 sec(-1), thus demonstrating that a free C-terminus is not required for activity. N- and O-glycanase treatment resulted in deglycosylation of all four proteins, and this caused a loss of xylosyltransferase activity for the C-terminally but not the N-terminally Flag-tagged proteins. The RGXT1 and RGXT2 proteins displayed an absolute requirement for Mn(2+) and were active over a broad pH range. Simple dialysis of media fractions or purification on phenyl Sepharose columns increased enzyme activities 2-8 fold enabling direct verification of the product formed in crude assay mixtures using electrospray ionization mass spectrometry. Pichia expressed and dialysed RGXT variants yielded activities within the range 0.011 to 0.013 U (1 U = 1 nmol conversion of substrate * min(-1) * microl medium(-1)) similar to those of RGXT1 and RGXT2 expressed in Baculovirus transfected insect Sf9 cells. In summary, the data presented suggest that Pichia is an attractive host candidate for expression of plant glycosyltransferases.
Petersen
Bent Larsen
BL
Cell Wall Biology and Molecular Plant Virology, Institute of Genetics and Biotechnology, Faculty of Agricultural Sciences, University of Aarhus and Centre for Pro-Active Plants (VKR), Thorvaldsensvej 40, Frederiksberg, 1871, Denmark. b.petersen@dias.kvl.dk
Egelund
Jack
J
Damager
Iben
I
Faber
Kirsten
K
Jensen
Jacob Krüger
JK
Yang
Zhang
Z
Bennett
Eric Paul
EP
Scheller
Henrik Vibe
HV
Ulvskov
Peter
P
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000166:cytology* / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D002473:Cell Wall / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D016695:Glycosyltransferases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D015151:Immunoblotting; D007313:Insecta / Q000166:cytology; D008969:Molecular Sequence Data; D010843:Pichia / Q000187:drug effects / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D029681:Arabidopsis Proteins; D002413:Cations, Divalent; D006023:Glycoproteins; D016695:Glycosyltransferases