Heterologous expression of plant cell wall glycosyltransferases in Pichia, pea and tobacco

Bent Larsen Petersen; Iben Damager; Kirsten Faber; Jack Egelund Madsen; Jacob Krüger Jensen; Merete T. Albrechtsen; Henrik Vibe Scheller; Peter Ulvskov

Heterologous expression of plant cell wall glycosyltransferases in Pichia, pea and tobacco

Bent Larsen Petersen, Iben Damager, Kirsten Faber, Jack Egelund Madsen, Jacob Krüger Jensen, Merete T. Albrechtsen, Henrik Vibe Scheller, Peter Ulvskov

Biomolecular Sciences

Publikation: Konferencebidrag › Poster › Forskning

Abstract

The plant cell wall (CW) consists of numerous complex and uniqe carbohydrate polymer

structures. Although the structure (sugar composition) of the various plant CW components

are known in some detail, functional characterisation of of the more than 300

glycosyltransferases (GTs), that are believed to participate in plant CW biosynthesis, has

been achieved in only a few cases. We have previously reported the characterisation of two

highly homologous plant-specific membrane-bound GTs, which when expressed as secreted

tagged soluble proteins in the baculo virus system, catalysed the transfer of xylose from

UDP-xylose on to the monosaccharide sugar fucose. Partly based on these data, the two

genes were proposed to function in the biosynthesis of pectic rhamnogalacturonan II (RG-II)

and designated RhamnoGalacturonan XylosylTransferase 1 and -2 (RGXT1 and -2),

accordingly (Egelund et al. 2006, The Plant Cell).

In the present study, Flag-tagged (MDYKDDDD) RGXT2 was expressed in Pichia pastoris as

secreted soluble protein, in pea (using the Pea early browning virus as expression vector) as

soluble intra-cellular protein and in tobacco as full length membrane bound protein. The

amount of expressed protein was estimated using anti Flag Ab and corresponding activity

monitored. Pros and cons of using the various expression systems are discussed.

Originalsprog	Engelsk
Publikationsdato	2007
Status	Udgivet - 2007
Begivenhed	Plant Biotech Denmark Annual Meeting - Copenhagen, Danmark Varighed: 23 jan. 2007 → 24 jan. 2007

Konference

Konference	Plant Biotech Denmark Annual Meeting
Land/Område	Danmark
By	Copenhagen
Periode	23/01/2007 → 24/01/2007

Bibliografisk note

Sider: 114

Adgang til dokumentet

http://www.plant-biotech.dk/meetings/abstraktbog2007.pdf

Citationsformater

Petersen, B. L., Damager, I., Faber, K., Madsen, J. E., Jensen, J. K., Albrechtsen, M. T., Scheller, H. V., & Ulvskov, P. (2007). Heterologous expression of plant cell wall glycosyltransferases in Pichia, pea and tobacco. Poster session præsenteret på Plant Biotech Denmark Annual Meeting, Copenhagen, Danmark. http://www.plant-biotech.dk/meetings/abstraktbog2007.pdf

@conference{9c868db0a02911dd86a6000ea68e967b,

title = "Heterologous expression of plant cell wall glycosyltransferases in Pichia, pea and tobacco",

abstract = "The plant cell wall (CW) consists of numerous complex and uniqe carbohydrate polymerstructures. Although the structure (sugar composition) of the various plant CW componentsare known in some detail, functional characterisation of of the more than 300glycosyltransferases (GTs), that are believed to participate in plant CW biosynthesis, hasbeen achieved in only a few cases. We have previously reported the characterisation of twohighly homologous plant-specific membrane-bound GTs, which when expressed as secretedtagged soluble proteins in the baculo virus system, catalysed the transfer of xylose fromUDP-xylose on to the monosaccharide sugar fucose. Partly based on these data, the twogenes were proposed to function in the biosynthesis of pectic rhamnogalacturonan II (RG-II)and designated RhamnoGalacturonan XylosylTransferase 1 and -2 (RGXT1 and -2),accordingly (Egelund et al. 2006, The Plant Cell).In the present study, Flag-tagged (MDYKDDDD) RGXT2 was expressed in Pichia pastoris assecreted soluble protein, in pea (using the Pea early browning virus as expression vector) assoluble intra-cellular protein and in tobacco as full length membrane bound protein. Theamount of expressed protein was estimated using anti Flag Ab and corresponding activitymonitored. Pros and cons of using the various expression systems are discussed.",

author = "Petersen, {Bent Larsen} and Iben Damager and Kirsten Faber and Madsen, {Jack Egelund} and Jensen, {Jacob Kr{\"u}ger} and Albrechtsen, {Merete T.} and Scheller, {Henrik Vibe} and Peter Ulvskov",

note = "Sider: 114; Plant Biotech Denmark Annual Meeting ; Conference date: 23-01-2007 Through 24-01-2007",

year = "2007",

language = "English",

}

TY - CONF

T1 - Heterologous expression of plant cell wall glycosyltransferases in Pichia, pea and tobacco

AU - Petersen, Bent Larsen

AU - Damager, Iben

AU - Faber, Kirsten

AU - Madsen, Jack Egelund

AU - Jensen, Jacob Krüger

AU - Albrechtsen, Merete T.

AU - Scheller, Henrik Vibe

AU - Ulvskov, Peter

N1 - Sider: 114

PY - 2007

Y1 - 2007

N2 - The plant cell wall (CW) consists of numerous complex and uniqe carbohydrate polymerstructures. Although the structure (sugar composition) of the various plant CW componentsare known in some detail, functional characterisation of of the more than 300glycosyltransferases (GTs), that are believed to participate in plant CW biosynthesis, hasbeen achieved in only a few cases. We have previously reported the characterisation of twohighly homologous plant-specific membrane-bound GTs, which when expressed as secretedtagged soluble proteins in the baculo virus system, catalysed the transfer of xylose fromUDP-xylose on to the monosaccharide sugar fucose. Partly based on these data, the twogenes were proposed to function in the biosynthesis of pectic rhamnogalacturonan II (RG-II)and designated RhamnoGalacturonan XylosylTransferase 1 and -2 (RGXT1 and -2),accordingly (Egelund et al. 2006, The Plant Cell).In the present study, Flag-tagged (MDYKDDDD) RGXT2 was expressed in Pichia pastoris assecreted soluble protein, in pea (using the Pea early browning virus as expression vector) assoluble intra-cellular protein and in tobacco as full length membrane bound protein. Theamount of expressed protein was estimated using anti Flag Ab and corresponding activitymonitored. Pros and cons of using the various expression systems are discussed.

AB - The plant cell wall (CW) consists of numerous complex and uniqe carbohydrate polymerstructures. Although the structure (sugar composition) of the various plant CW componentsare known in some detail, functional characterisation of of the more than 300glycosyltransferases (GTs), that are believed to participate in plant CW biosynthesis, hasbeen achieved in only a few cases. We have previously reported the characterisation of twohighly homologous plant-specific membrane-bound GTs, which when expressed as secretedtagged soluble proteins in the baculo virus system, catalysed the transfer of xylose fromUDP-xylose on to the monosaccharide sugar fucose. Partly based on these data, the twogenes were proposed to function in the biosynthesis of pectic rhamnogalacturonan II (RG-II)and designated RhamnoGalacturonan XylosylTransferase 1 and -2 (RGXT1 and -2),accordingly (Egelund et al. 2006, The Plant Cell).In the present study, Flag-tagged (MDYKDDDD) RGXT2 was expressed in Pichia pastoris assecreted soluble protein, in pea (using the Pea early browning virus as expression vector) assoluble intra-cellular protein and in tobacco as full length membrane bound protein. Theamount of expressed protein was estimated using anti Flag Ab and corresponding activitymonitored. Pros and cons of using the various expression systems are discussed.

M3 - Poster

T2 - Plant Biotech Denmark Annual Meeting

Y2 - 23 January 2007 through 24 January 2007

ER -