Expression system for enhanced green fluorescence protein conjugated recombinant antibody fragment.
Recent development of recombinant antibody technology has enabled fusion of recombinant antibody fragment with fluorescent proteins for various applications such as flow cytometry, fluorescence immunoassay, and fluorescent microscopy. In this study, we generated various forms of green fluorescence protein (EGFP)-fused anti-c-Met antibody fragment. Among these fusion proteins, EGFP fusion to the light chain showed high expression in a soluble form of protein in E. coli, and high binding activity to c-Met. A feasibility of the constructs was further examined by replacing the Fab gene by a Fab library of catalytic subunit of protein kinase A (PKA) to construct the Fab library in EGFP fused form. We also constructed the conventional Fab library. After a series of biopanning, we found that the binding capability of EGFP-anti-PKA Fab was comparable with anti-PKA Fab. Sequence analysis of the selected clones showed > or =99% identity in amino acid sequence and shared the same CDR sequence. These results demonstrate that EGFP fusion to the light chain using our vector system does not influence the selection of reactive Fab and that this vector system is useful for EGFP fusion to Fab to develop a one-step detection system.
Yi
Kye Sook
KS
Department of Biochemistry and Institute of Cardiovascular Research, Chonbuk National University Medical School, Chonju, Korea.
Chung
Junho
J
Park
Kwang-Hyun
KH
Kim
Kisu
K
Im
Shin-Young
SY
Choi
Cha-Yong
CY
Im
Mie-Jae
MJ
Kim
Uh-Hyun
UH
D000595:Amino Acid Sequence; D015870:Gene Expression / Q000276:immunology / Q000502:physiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins