A new gibberellin detection system in living cells based on antibody V(H)/V(L) interaction.
As a new detection method of bioactive gibberellin A(4) (GA(4)) in living cells, a combined system of GA(4)-dependent interaction of V(H) and V(L) composed of a variable region fragment (Fv) of anti-GA(4) antibodies and protein-fragment complementation assay (PCA) was developed. First, when V(H) and V(L) were displayed in proximity on a phage, they could constitute a functional Fv. Thereafter, V(H) and V(L) were shown to interact with each other in a GA(4)-dependent manner. We then applied this interaction to PCA using GFP as a reporter. V(H) fused to the C-terminal half of GFP and V(L) fused to the N-terminal half of GFP were simultaneously expressed in Escherichia coli. The E. coli in which these fusion proteins were inductively produced in the presence of GA(4) showed clear GFP fluorescence, while those in the absence of GA(4) showed only scarce GFP fluorescence, demonstrating the feasibility of this system to detect GA(4) in living organisms.
Lee
Younggiu
Y
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Asami
Tadao
T
Yamaguchi
Isomaro
I
Ueda
Hiroshi
H
Suzuki
Yoshihito
Y
D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005875:Gibberellins / Q000032:analysis* / Q000276:immunology; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology*; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D005875:Gibberellins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C532593:gibberellin A4; D049452:Green Fluorescent Proteins