异源表达植物蛋白的糖基转移酶活性的体外检测

Carsten P Ade; Felix Bemm; James M J Dickson; Christian Walter; Philip J Harris

doi:10.21769/BioProtoc.1285

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Peer-reviewed

In vitro Assay of the Glycosyltransferase Activity of a Heterologously Expressed Plant Protein

异源表达植物蛋白的糖基转移酶活性的体外检测

PH Philip J Harris email

发布: 2014年11月05日第4卷第21期 DOI: 10.21769/BioProtoc.1285 浏览次数: 11849

评审: Tie LiuAnonymous reviewer(s)

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Cover of The Plant Journal, featuring study using the protocol.

Apr 2014

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Abstract

Glycosyltransferases are carbohydrate active enzymes containing catalytic modules involved in catalysing the biosynthesis of glycosidic bonds in oligo- and polysaccharides and glycoconjugates. One of the most comprehensive collections of Carbohydrate Active enZYmes is the CAZy database (http://www.cazy.org) comprising 120,000 glycosyltransferases allocated to 96 families based mainly on sequence homologies of their conserved and catalytically active domains (Cantarel et al., 2009). Interestingly, the glycosyltransferase activities of only about 1.6% of these proteins have been experimentally characterized (Lombard et al., 2014). In recent years, membrane-bound glycosyltransferases of a number of families have been shown to play a key role in the biosynthesis of plant cell-wall polysaccharides (Doblin et al., 2010; Scheller and Ulvskov, 2010; Driouich et al., 2012). They catalyze the transfer of glycosyl residues from donor nucleotide sugars to acceptors, forming the glycosidic bonds between adjacent glycosyl residues. Family 34 contains glycosyltransferases that have been shown to be involved in the biosynthesis of xyloglucans and transfer xylosyl residues to (1→4)-β-glucan chains (Keegstra and Cavalier, 2011). Our previous work suggests that Pinus radiata protein PrGT34B is a xyloglucan (1→6)-α-xylosyltransferase (Ade et al., 2014). Here, we describe a procedure for determining the xylosyltransferase activity of PrGT34B in vitro. We measured the transfer of xylose from the donor substrate UDP-xylose to different cello-oligosaccharide acceptor substrates under controlled reaction conditions. The assays include quantification of radioactively labeled reaction products and their identification by mass spectrometry. We also describe the purification, identification and quantification of the heterologously expressed recombinant protein PrGT34B in preparation for its use in the assays. This procedure may be applied to a wide range of glycosyltransferases in many different plant species.

Materials and Reagents

Spodoptera frugiperda cells (Sf9) (Life Technologies, Invitrogen^TM, catalog number: 11496-015 )
Vector pFastBacHTa^TM (Life Technologies, Invitrogen^TM, catalog number: 10584-027 )
Escherichia coli (E. coli) strains DH10Bac (Life Technologies, Invitrogen^TM, catalog number: 10359-016 )
20% ethanol
SDS
β-mercaptoethanol
Glycerol
Bromophenol blue
HEPES-NaOH (pH 7.0)
MgCl₂
MnCl₂
Triton X-100
Benzonase^® nuclease (Novagen, catalog number: 70746 )
Protease inhibitors (complete EDTA free, protease inhibitor cocktail tablet) (Roche Diagnostics, catalog number: 0 4693159001 )
Imidazole
Cellotetraose, cellopentaose and cellohexaose (Associates of Cape Cod, Northstar BioProducts^®, catalog numbers: 400402-1 , 400404-1 , 400406-1 )
Cellobiose (Sigma-Aldrich, catalog number: C7252 )
UDP-D-xylose (CarboSource Services, Complex Carbohydrate Research Center, product code: UDP-Xylose )
UDP-[¹⁴C-U]xylose (151.8 mCi/mmol) (PerkinElmer, catalog number: NEC543005UC )
TALON metal affinity resin (Takara Bio Company, Clontech, catalog number: 635501 )
BenchMark His-tagged protein standard (Life Technologies, Invitrogen^TM, catalog number: LC5606 )
Precision Plus protein unstained standard (Bio-Rad Laboratories, catalog number: 161-0363 )
Mouse anti-His₆ monoclonal antibody (GE Healthcare, product code: 27-4710-01 )
Horseradish peroxidase (HRP) conjugated secondary sheep anti-mouse antibody (GE Healthcare, product code: NA931-100UL )
SYPRO^® Ruby protein gel stain (Life Technologies, Molecular Probes^®, catalog number: S-21900 )
Ion exchange resin Dowex 1x8-200 (chloride form) (Sigma-Aldrich, catalog number: 217425 )
2,5-Dihydroxybenzoic acid (Sigma-Aldrich, catalog number: 85707 )
Starscint high flash-point LSC-cocktail (PerkinElmer)
PAGE loading dye (see Recipes)
Hypotonic lysis buffer (see Recipes)
Equilibration/wash buffer for IMAC (see Recipes)
Sample preparation buffer for IMAC (see Recipes)
IMAC elution buffer (see Recipes)
Reaction buffer for enzymatic assays (with radioactively labeled UDP-xylose) (see Recipes)
Reaction buffer for enzymatic assays (without radioactively labeled UDP-xylose) (see Recipes)

Equipment

Empty gravity-flow chromatography columns (20 ml volume) (Bio-Rad Laboratories, catalog number: 732-1010 )
Micro Bio-Spin chromatography columns (Bio-Rad Laboratories, catalog number: 732-6204 )
Amersham ECL detection system (GE Healthcare, product code: RPN2105 )
PVDF transfer membrane (Amersham Hybond-P) (GE Healthcare, product code: 10600023 )
NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific)
LAS-3000 Lite imager with software LAS-3000 Image Reader (Fujifilm Corporation)
FLA 2000 phosphorimager with software FLA 2000G (Fujifilm Corporation)
Rackbeta LKB Wallac (PerkinElmer, model: 1209 )
Voyager DE^TM PRO MALDI-TOF Workstation (Life Technologies, Applied Biosystems^®)
Note: We believe that the reproducibility of the experiments does not depend on the precise equipment stated above but can be achieved using any other equivalent instrumentation.

Software

Software Multi Gauge v2.2 (Fujifilm Corporation)
Data Explorer (version 4.0.0.0) (Life Technologies, Applied Biosystems^®)
Software FLA 2000G (Fujifilm Corporation)
Software LAS-3000 Image Reader (Fujifilm Corporation)

Procedure

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Ade, C. P., Bemm, F., Dickson, J. M. J., Walter, C. and Harris, P. J. (2014). In vitro Assay of the Glycosyltransferase Activity of a Heterologously Expressed Plant Protein. Bio-protocol 4(21): e1285. DOI: 10.21769/BioProtoc.1285.