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Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana

Dominique Eeckhout UGent, Esbjörn Fiers UGent, Rebecca Sienaert, Veerle Snoeck, Anna Depicker UGent and Geert De Jaeger UGent (2000) EUROPEAN JOURNAL OF BIOCHEMISTRY. 267(23). p.6775-6783
abstract
In order to obtain recombinant antibody fragments that bind the cell-cycle protein CDC2a from Arabidopsis thaliana (CDC2aAt), two phage display libraries of single-chain variable (scFv) fragments were constructed. One library was derived from mice immunized with recombinant CDC2aAt N-terminally fused to a His(6)-tag (His-CDC2aAt) and the other was made out of an anti-PSTAIRE hybridoma cell line. Six specific His-CDC2aAt-binding phage clones (3D1, 3D2, 3D10, 3D25, 4D21 and 4D47) were isolated by panning. The isolated monoclonal phage clones, as well as the soluble scFv fragments produced in the periplasm of Escherichia coli, bind His-CDC2aAt in ELISA and on Western blots. Moreover, four clones (3D1, 3D2, 3D10 and 4D21) detect specifically CDC2aAt from Arabidopsis cell suspensions on Western blots. Clone 4D21 binds the PSTAIRE epitope, whereas the 3D1, 3D2 and 3D10 clones bind, as yet unidentified, epitopes of CDC2aAt. Furthermore, the accumulation and antigen-binding activity of these scFv fragments in a reducing environment were assessed. No interaction could be shown between the scFv fragments and CDC2aAt in a yeast two-hybrid assay. However, after transient expression of the scFv fragments in the cytosol of tobacco leaves, three of six scFv fragments (3D1, 3D2 and 3D10) accumulated in the plant cytosol and ELISA results indicate that these scFv fragments retained antigen-binding activity.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
phage display, cyclin-dependent kinase, PSTAIRE motif, single-chain variable fragment, CHAIN VARIABLE FRAGMENTS, CYCLIN-DEPENDENT KINASES, PETUNIA-HYBRIDA, PROTEIN-KINASE, PHAGE DISPLAY, CELL-DIVISION, EXPRESSION, CYTOSOL, SEQUENCE, BINDING
journal title
EUROPEAN JOURNAL OF BIOCHEMISTRY
Eur. J. Biochem.
volume
267
issue
23
pages
6775 - 6783
Web of Science type
Article
Web of Science id
000165457100006
ISSN
0014-2956
DOI
10.1046/j.1432-1327.2000.01770.x
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
124913
handle
http://hdl.handle.net/1854/LU-124913
date created
2004-01-14 13:36:00
date last changed
2016-12-19 15:37:49
@article{124913,
  abstract     = {In order to obtain recombinant antibody fragments that bind the cell-cycle protein CDC2a from Arabidopsis thaliana (CDC2aAt), two phage display libraries of single-chain variable (scFv) fragments were constructed. One library was derived from mice immunized with recombinant CDC2aAt N-terminally fused to a His(6)-tag (His-CDC2aAt) and the other was made out of an anti-PSTAIRE hybridoma cell line. Six specific His-CDC2aAt-binding phage clones (3D1, 3D2, 3D10, 3D25, 4D21 and 4D47) were isolated by panning. The isolated monoclonal phage clones, as well as the soluble scFv fragments produced in the periplasm of Escherichia coli, bind His-CDC2aAt in ELISA and on Western blots. Moreover, four clones (3D1, 3D2, 3D10 and 4D21) detect specifically CDC2aAt from Arabidopsis cell suspensions on Western blots. Clone 4D21 binds the PSTAIRE epitope, whereas the 3D1, 3D2 and 3D10 clones bind, as yet unidentified, epitopes of CDC2aAt. Furthermore, the accumulation and antigen-binding activity of these scFv fragments in a reducing environment were assessed. No interaction could be shown between the scFv fragments and CDC2aAt in a yeast two-hybrid assay. However, after transient expression of the scFv fragments in the cytosol of tobacco leaves, three of six scFv fragments (3D1, 3D2 and 3D10) accumulated in the plant cytosol and ELISA results indicate that these scFv fragments retained antigen-binding activity.},
  author       = {Eeckhout, Dominique and Fiers, Esbj{\"o}rn and Sienaert, Rebecca and Snoeck, Veerle and Depicker, Anna and De Jaeger, Geert},
  issn         = {0014-2956},
  journal      = {EUROPEAN JOURNAL OF BIOCHEMISTRY},
  keyword      = {phage display,cyclin-dependent kinase,PSTAIRE motif,single-chain variable fragment,CHAIN VARIABLE FRAGMENTS,CYCLIN-DEPENDENT KINASES,PETUNIA-HYBRIDA,PROTEIN-KINASE,PHAGE DISPLAY,CELL-DIVISION,EXPRESSION,CYTOSOL,SEQUENCE,BINDING},
  language     = {eng},
  number       = {23},
  pages        = {6775--6783},
  title        = {Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana},
  url          = {http://dx.doi.org/10.1046/j.1432-1327.2000.01770.x},
  volume       = {267},
  year         = {2000},
}

Chicago
Eeckhout, Dominique, Esbjörn Fiers, Rebecca Sienaert, Veerle Snoeck, Anna Depicker, and Geert De Jaeger. 2000. “Isolation and Characterization of Recombinant Antibody Fragments Against CDC2a from Arabidopsis Thaliana.” European Journal of Biochemistry 267 (23): 6775–6783.
APA
Eeckhout, D., Fiers, E., Sienaert, R., Snoeck, V., Depicker, A., & De Jaeger, G. (2000). Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana. EUROPEAN JOURNAL OF BIOCHEMISTRY, 267(23), 6775–6783.
Vancouver
1.
Eeckhout D, Fiers E, Sienaert R, Snoeck V, Depicker A, De Jaeger G. Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana. EUROPEAN JOURNAL OF BIOCHEMISTRY. 2000;267(23):6775–83.
MLA
Eeckhout, Dominique, Esbjörn Fiers, Rebecca Sienaert, et al. “Isolation and Characterization of Recombinant Antibody Fragments Against CDC2a from Arabidopsis Thaliana.” EUROPEAN JOURNAL OF BIOCHEMISTRY 267.23 (2000): 6775–6783. Print.