Overexpression and Functional Stabilization of Recombinant Human Lysophosphatidic Acid Receptor 1 Using an Amphiphatic Polymer

Han, Seong‐Gu; Baek, Seung-Il; Lee, Won Kyu; Padmanaban, Sudakar; Yu, Yeon Gyu

doi:10.1002/bkcs.11048

Cited by 9 publications

(16 citation statements)

References 37 publications

(58 reference statements)

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“…The fusion protein (P9‐LPA 1 ) was solubilized with sarkosyl and purified to more than 95% homogeneity (Figure (a)). The purified p9‐LPA1 showed LPA‐dependent interaction with the G αi as previously described . The purified P9‐LPA 1 was also reconstituted in a disc‐shaped lipid bilayer structure, called a nanodisc, using MSP1.…”

Section: Resultsmentioning

confidence: 61%

“…For scFv screening, LPA 1 must be in its native conformation. As previously described, LPA 1 was expressed as a P9 fusion protein in the membrane fraction of E. coli cells . The fusion protein (P9‐LPA 1 ) was solubilized with sarkosyl and purified to more than 95% homogeneity (Figure (a)).…”

Section: Resultsmentioning

confidence: 99%

“…A human LPA 1 expression vector (pP9‐LPA 1 ) was prepared as previously described . This vector expressed an LPA 1 fusion protein (P9‐LPA 1 ), which consisted of an N‐terminus P9 domain and the coding region of LPA 1 , in the periplasmic membrane of E. coli .…”

Section: Methodsmentioning

confidence: 99%

“…However, the development of GPCR‐specific antibodies has been hindered by a lack of purified active GPCRs. Previously, we successfully purified LPA 1 and LPA 2 as fusion proteins with the P9 domain, and the purified LPA 1 and LPA 2 stabilized in detergents or an amphiphilic polymer showed a ligand‐dependent specific interaction with the alpha subunit of G i3 protein . The purified LPA 2 can be used to isolate single‐chain antibodies (scFvs) from M13 phage library displaying scFv domain .…”

Section: Introductionmentioning

confidence: 97%

“…The purified LPA 2 can be used to isolate single‐chain antibodies (scFvs) from M13 phage library displaying scFv domain . Although LPA 1 stabilized by an amphiphilic polymer showed ligand binding capacity, the native structure of LPA 1 might be preserved in a lipid bilayer environment. A nanodisc structure consisting of a 10–14 nm lipid‐bilayer surrounded by lipid‐stabilizing proteins, such as MSP1, has been developed and applied to stabilize membrane proteins, including GPCRs …”

Section: Introductionmentioning

confidence: 99%

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Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPA₁) from a M13 Phage Display Library Using Purified LPA₁ Stabilized in Nanodiscs

Jung

Han

et al. 2019

Bulletin Korean Chem Soc

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G‐protein coupled receptors (GPCRs) comprise the largest membrane protein family and are involved in various kinds of physiological phenomena. LPA1 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, LPA1‐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 LPA1 using a purified LPA1 in nanodiscs and a library of M13 phages displaying human naïve single‐chain variable fragment (scFv) sequences. The purified P9‐LPA1 was stabilized in native conformation in nanodiscs and attached to immobilized Gαi3 protein, and then M13 phages specific to LPA1 were isolated after several rounds of biopanning. Two clones which specifically interacted with the immobilized LPA1 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 LPA1 with KD values of 300–400 nM. When LPA1‐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 LPA1 agonist.

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Section: Resultsmentioning

confidence: 61%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPA₁) from a M13 Phage Display Library Using Purified LPA₁ Stabilized in Nanodiscs

Jung

Han

et al. 2019

Bulletin Korean Chem Soc

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Use of a Poly‐γ‐Glutamic Acid‐Derived Amphipathic Polypeptide for the Reconstitution of Membrane Proteins

Han

Choi

Kim

et al. 2020

Bulletin Korean Chem Soc

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A robust antibody discovery platform for difficult‐to‐express G protein‐coupled receptors

et al. 2022

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G protein-coupled receptors (GPCRs) are in the spotlight as drug targets due to the fact that multiple research results have verified the correlation between the activation of GPCRs and disease indications. This is because the GPCRs are present across the cell membranes, which interact with either extracellular ligands or other types of compartments and simultaneously mediate intracellular signaling. Despite the importance of the GPCRs as drug targets, they are too difficult to express in soluble forms. Currently, the difficulty of preparing functional GPCRs and the lack of efficient antibody screening methods are the most challenging steps in the discovery of antibodies targeting GPCRs. In this study, we developed a powerful platform that facilitates isolating GPCRspecific antibodies by obviating difficulties in GPCR preparation. The strategies include (i) conjugation of the P9 peptide, an envelope protein of Pseudomonas phi6, to the N-terminus of GPCRs to improve the expression level of the GPCRs in Escherichia coli, (ii) stabilization of the GPCRs in their active forms with amphiphilic poly-γ-glutamate (APG) to shield the seven hydrophobic transmembrane domains, and (iii) further limiting the size of the APG complex to improve the chance to isolate antibodies targeting the proteins-of-interest. Capitalizing on the above strategies, we could prepare GPCR proteins in their active forms as facile as other general-soluble antigen proteins. Furthermore, this protocol was validated to be successful in discovering three individual GPCR-specific antibodies targeting glucagon-like peptide-1 receptor, C-X-C chemokine receptor type 4, and prostaglandin E2 receptor 4 in this study. K E Y W O R D S amphiphilic poly-γ-glutamate, antibody discovery, G protein-coupled receptors, phage display, purification 1 | INTRODUCTION G protein-coupled receptors (GPCRs) are one of the membrane receptor proteins that generate intracellular signal transduction in response to various extracellular signals. The cell signaling by GPCRs is generally mediated through ligand-induced activation, which makes changes in the structure of the GPCRs and activates proteins that interact with their intracellular domains inside the cell, triggering multiple intracellular events such as Nam Hyuk Kim and Sumin Kang contributed equally to this work.

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Overexpression and Functional Stabilization of Recombinant Human Lysophosphatidic Acid Receptor 1 Using an Amphiphatic Polymer

Cited by 9 publications

References 37 publications

Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPA₁) from a M13 Phage Display Library Using Purified LPA₁ Stabilized in Nanodiscs

Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPA₁) from a M13 Phage Display Library Using Purified LPA₁ Stabilized in Nanodiscs

Use of a Poly‐γ‐Glutamic Acid‐Derived Amphipathic Polypeptide for the Reconstitution of Membrane Proteins

A robust antibody discovery platform for difficult‐to‐express G protein‐coupled receptors

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Overexpression and Functional Stabilization of Recombinant Human Lysophosphatidic Acid Receptor 1 Using an Amphiphatic Polymer

Cited by 9 publications

References 37 publications

Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPA1) from a M13 Phage Display Library Using Purified LPA1 Stabilized in Nanodiscs

Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPA1) from a M13 Phage Display Library Using Purified LPA1 Stabilized in Nanodiscs

Use of a Poly‐γ‐Glutamic Acid‐Derived Amphipathic Polypeptide for the Reconstitution of Membrane Proteins

A robust antibody discovery platform for difficult‐to‐express G protein‐coupled receptors

Contact Info

Product

Resources

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Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPA₁) from a M13 Phage Display Library Using Purified LPA₁ Stabilized in Nanodiscs

Isolation of Single Chain Antibodies Specific to Lysophosphatidic Acid Receptor 1 (LPA₁) from a M13 Phage Display Library Using Purified LPA₁ Stabilized in Nanodiscs