Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples. We proposed here an efficient in vitro method to produce [15N, 13C, 2H]-labeled Fabs enabling high resolution NMR investigations of these powerful therapeutics. As an open system, the cell-free expression mode enables fine-tuned control of the redox potential in presence of disulfide bond isomerase to enhance the formation of native disulfide bonds. Moreover, inhibition of transaminases in the S30 cell-free extract offers the opportunity to produce perdeuterated Fab samples directly in 1H2O medium, without the need for a time-consuming and inefficient refolding process. This specific protocol was applied to produce an optimally labeled sample of a therapeutic Fab, enabling the sequential assignment of 1HN, 15N, 13Câ?², 13Cα, 13Cβ resonances of a full-length Fab. 90% of the backbone resonances of a Fab domain directed against the human LAMP1 glycoprotein were assigned successfully, opening new opportunities to study, at atomic resolution, Fabsâ?? higher order structures, dynamics and interactions, using solution-state NMR.
[NMR paper] Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins.
Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins.
Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins.
Biomolecules. 2020 Oct 19;10(10):
Authors: Morató A, Elena-Real CA, Popovic M, Fournet A, Zhang K, Allemand F, Sibille N, Urbanek A, Bernadó P
Abstract
The high-resolution structural...
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[NMR paper] P-B desulfurization: an enabling method for protein chemical synthesis and site-specific deuteration
P-B desulfurization: an enabling method for protein chemical synthesis and site-specific deuteration
Native chemical ligation has provided a powerful tool for protein chemical synthesis. Herein, we report an unprecedented mild system (TCEP-NaBH4 or TCEP-LiBEt3H) for chemoselective peptide desulfurization to achieve effective protein synthesis via the native chemical ligation-desulfurization approach. This method, termed P-B desulfurization, features usage of common reagents, simplicity of operation, robustness, high yielding, clean conversion and versatile functionality compatibility...
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[NMR paper] Application of Site-Specific Spin Labeling for NMR Detecting Inhibitor-Induced Conformational Change of HIV-1 Reverse Transcriptase.
Application of Site-Specific Spin Labeling for NMR Detecting Inhibitor-Induced Conformational Change of HIV-1 Reverse Transcriptase.
Related Articles Application of Site-Specific Spin Labeling for NMR Detecting Inhibitor-Induced Conformational Change of HIV-1 Reverse Transcriptase.
ChemMedChem. 2016 Jan 25;
Authors: Seetaha S, Yagi-Utsumi M, Yamaguchi T, Ishii K, Hannongbua S, Choowongkomon K, Kato K
Abstract
Paramagnetism-assisted nuclear magnetic resonance (NMR) techniques can provide long-range structural information...
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01-26-2016 03:40 PM
[NMR paper] Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins.
Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins.
Related Articles Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins.
Curr Opin Struct Biol. 2015 Apr 13;32:113-122
Authors: Kerfah R, Plevin MJ, Sounier R, Gans P, Boisbouvier J
Abstract
Nuclear magnetic resonance (NMR) spectroscopy is a uniquely powerful tool for studying the structure, dynamics and interactions of biomolecules at atomic resolution. In the past 15 years, the...
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Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins
Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins
Publication date: June 2015
Source:Current Opinion in Structural Biology, Volume 32</br>
Author(s): Rime Kerfah , Michael J Plevin , Remy Sounier , Pierre Gans , Jerome Boisbouvier</br>
Nuclear magnetic resonance (NMR) spectroscopy is a uniquely powerful tool for studying the structure, dynamics and interactions of biomolecules at atomic resolution. In the past 15 years, the development of new isotopic labeling strategies has opened the possibility of exploiting...
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04-14-2015 01:24 PM
[NMR paper] Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.
Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.
Preparation of Multiple Site-Specific Mutant Proteins for NMR Studies by PCR-Directed Cell-Free Protein Synthesis.
Methods Mol Biol. 2014;1118:169-87
Authors: Ozawa K, Qi R
Abstract
Cell-free protein synthesis (CFPS) offers a fast and inexpensive approach to selectively label proteins with isotopes that can then be detected by nuclear magnetic resonance (NMR) spectroscopy directly in the translation mixture. We describe...
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[NMR paper] Cell-free protein production and labeling protocol for NMR-based structural proteomic
Cell-free protein production and labeling protocol for NMR-based structural proteomics.
Related Articles Cell-free protein production and labeling protocol for NMR-based structural proteomics.
Nat Methods. 2004 Nov;1(2):149-53
Authors: Vinarov DA, Lytle BL, Peterson FC, Tyler EM, Volkman BF, Markley JL
Structural proteomics requires robust, scalable methods. Here we describe a wheat germ cell-free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell-based methods....
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[NMR paper] Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and ir
Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.
Related Articles Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli.
J Biomol NMR. 2000 Aug;17(4):311-22
Authors: Sorkin DL, Miller AF
We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide...
Enabling site-specific NMR investigations of therapeutic FabÂ*using a cell-free based isotopic labeling approach:Â*application to anti-LAMP1 Fab
Linear Mode
