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Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris

Bassam Al Atalah UGent, Elke Fouquaert UGent, Dieter Vanderschaeghe UGent, Paul Proost, Jan Balzarini, David F Smith, Pierre Rouge, Yi Lasanajak, Nico Callewaert UGent and Els Van Damme UGent (2011) FEBS JOURNAL. 278(12). p.2064-2079
abstract
The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces alpha-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
GALANTHUS-NIVALIS AGGLUTININ, JACALIN-RELATED LECTINS, LARGE-SCALE PRODUCTION, HETEROLOGOUS PROTEINS, BINDING SPECIFICITY, GALACTOSE-BINDING, CALYSTEGIA-SEPIUM, MOLECULAR-CLONING, TOBACCO LECTIN, HIGH-MANNOSE, antiviral activity, glycan array, lectin, nucleus, Orysata
journal title
FEBS JOURNAL
FEBS J.
volume
278
issue
12
pages
2064 - 2079
Web of Science type
Article
Web of Science id
000291242700011
JCR category
BIOCHEMISTRY & MOLECULAR BIOLOGY
JCR impact factor
3.79 (2011)
JCR rank
89/286 (2011)
JCR quartile
2 (2011)
ISSN
1742-464X
DOI
10.1111/j.1742-4658.2011.08122.x
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
1269239
handle
http://hdl.handle.net/1854/LU-1269239
date created
2011-06-20 15:53:28
date last changed
2012-06-26 14:32:20
@article{1269239,
  abstract     = {The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces alpha-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.},
  author       = {Al Atalah, Bassam and Fouquaert, Elke and Vanderschaeghe, Dieter and Proost, Paul and Balzarini, Jan and Smith, David F and Rouge, Pierre and Lasanajak, Yi and Callewaert, Nico and Van Damme, Els},
  issn         = {1742-464X},
  journal      = {FEBS JOURNAL},
  keyword      = {GALANTHUS-NIVALIS AGGLUTININ,JACALIN-RELATED LECTINS,LARGE-SCALE PRODUCTION,HETEROLOGOUS PROTEINS,BINDING SPECIFICITY,GALACTOSE-BINDING,CALYSTEGIA-SEPIUM,MOLECULAR-CLONING,TOBACCO LECTIN,HIGH-MANNOSE,antiviral activity,glycan array,lectin,nucleus,Orysata},
  language     = {eng},
  number       = {12},
  pages        = {2064--2079},
  title        = {Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris},
  url          = {http://dx.doi.org/10.1111/j.1742-4658.2011.08122.x},
  volume       = {278},
  year         = {2011},
}

Chicago
Al Atalah, Bassam, Elke Fouquaert, Dieter Vanderschaeghe, Paul Proost, Jan Balzarini, David F Smith, Pierre Rouge, Yi Lasanajak, Nico Callewaert, and Els Van Damme. 2011. “Expression Analysis of the Nucleocytoplasmic Lectin ‘Orysata’ from Rice in Pichia Pastoris.” Febs Journal 278 (12): 2064–2079.
APA
Al Atalah, B., Fouquaert, E., Vanderschaeghe, D., Proost, P., Balzarini, J., Smith, D. F., Rouge, P., et al. (2011). Expression analysis of the nucleocytoplasmic lectin “Orysata” from rice in Pichia pastoris. FEBS JOURNAL, 278(12), 2064–2079.
Vancouver
1.
Al Atalah B, Fouquaert E, Vanderschaeghe D, Proost P, Balzarini J, Smith DF, et al. Expression analysis of the nucleocytoplasmic lectin “Orysata” from rice in Pichia pastoris. FEBS JOURNAL. 2011;278(12):2064–79.
MLA
Al Atalah, Bassam, Elke Fouquaert, Dieter Vanderschaeghe, et al. “Expression Analysis of the Nucleocytoplasmic Lectin ‘Orysata’ from Rice in Pichia Pastoris.” FEBS JOURNAL 278.12 (2011): 2064–2079. Print.