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Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPA 1 ) from a M13 Phage Display Library Using Purified LPA 1 Stabilized in Nanodiscs
Author(s) -
Jung Ji Hae,
Han SeongGu,
Ju ManSeok,
Jung Sang Taek,
Yu Yeon Gyu
Publication year - 2019
Publication title -
bulletin of the korean chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.237
H-Index - 59
ISSN - 1229-5949
DOI - 10.1002/bkcs.11751
Subject(s) - lysophosphatidic acid , phage display , antibody , immunoglobulin light chain , microbiology and biotechnology , receptor , chemistry , g protein coupled receptor , biology , biochemistry , peptide , immunology
G‐protein coupled receptors (GPCRs) comprise the largest membrane protein family and are involved in various kinds of physiological phenomena. LPA 1 belongs to the rhodopsin‐type GPCR family and mediates various biological functions, such as cell proliferation, platelet aggregation, smooth muscle contraction, and tumor cell invasion. Hence, LPA 1 ‐specific antibodies have the potential to be used as therapeutic agents against cancer or ophthalmic disease. In this study, we identified single‐chain antibodies specific to LPA 1 using a purified LPA 1 in nanodiscs and a library of M13 phages displaying human naïve single‐chain variable fragment (scFv) sequences. The purified P9‐LPA 1 was stabilized in native conformation in nanodiscs and attached to immobilized G αi3 protein, and then M13 phages specific to LPA 1 were isolated after several rounds of biopanning. Two clones which specifically interacted with the immobilized LPA 1 were isolated, and single‐chain antibody fragments (scAbs) that contained the isolated scFv fragment and a human kappa light chain constant domain were constructed and expressed in E. coli . The two purified scAbs (B8 and D4) showed specific binding to LPA 1 with K D values of 300–400 nM. When LPA 1 ‐overexpressing HT29 cells were treated with a scAb (D4) and lysophosphatidic acid, an increase in the cytosolic calcium level was observed relative to cells treated only with lysophosphatidic acid, indicating that the isolated single chain antibody (D4) acts as a functional LPA 1 agonist.