Rapid isolation of intrabody candidates by using an optimized non-immune phage antibody library.
Phage antibody library is a promising tool for rapidly creating in vitro single-chain Fv (scFv) antibodies to various antigens. The scFv can also act like a subcellularly-expressed antibody, known as intrabody, and can either be used as a novel research tool or used efficiently for targeted molecular therapy. However, there are only a few existing reports about the successful expression of scFvs as functional antibodies in the cell, mainly because poor quality scFv phage antibody libraries were used to isolate the intrabody clones. The aim of this study was to isolate intrabody-forming scFv clones from the nonimmune scFv phage antibody library we have generated. Using this library, we isolated a scFv clone against the apoptosis-related intracellular protein Bid in two weeks. To evaluate the intrabody-forming quality of this anti-Bid scFv clone, we expressed it in cultured mammalian cells after fusing it with the fluorescent protein Venus. The expression of the soluble form of anti-Bid scFv-Venus fusion protein was confirmed by fluorescence microscopy analysis. These results show that our scFv phage library is not only optimized for antibody production but can also be used to efficiently generate intrabodies.
Laboratory of Pharmaceutical Proteomics, Osaka University, Osaka, Japan.
K
K
Nagano
S
S
Imai
Y
Y
Mukai
S
S
Nakagawa
Y
Y
Abe
H
H
Kamada
S
S
Tsunoda
Y
Y
Tsutsumi
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry*; D019138:Antibodies, Blocking / Q000737:chemistry / Q000494:pharmacology; D000918:Antibody Specificity; D051023:BH3 Interacting Domain Death Agonist Protein / Q000276:immunology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology
D000906:Antibodies; D019138:Antibodies, Blocking; D051023:BH3 Interacting Domain Death Agonist Protein; D019151:Peptide Library; D011993:Recombinant Fusion Proteins