Isolation and characterization of recombinant antibody fragments against CDC2a from Arabidopsis thaliana.
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 His6-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.
Eeckhout
D
D
Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie, Universiteit Gent, Belgium.
Fiers
E
E
Sienaert
R
R
Snoeck
V
V
Depicker
A
A
De Jaeger
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D017360:Arabidopsis / Q000737:chemistry* / Q000276:immunology*; D029681:Arabidopsis Proteins; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000737:chemistry / Q000276:immunology*; D018388:Codon, Terminator; D018844:Cyclin-Dependent Kinases / Q000737:chemistry* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D004926:Escherichia coli / Q000378:metabolism; D006825:Hybridomas; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D000911:Antibodies, Monoclonal; D029681:Arabidopsis Proteins; D018388:Codon, Terminator; D000939:Epitopes; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D016203:CDC2 Protein Kinase; C418966:CDC2A protein, Arabidopsis; D018844:Cyclin-Dependent Kinases