Generation of Recombinant Antibody Fragments for Membrane Protein Crystallization

Mir, Syed; Escher, Claudia; Kao, Wei-Chun; Birth, Dominic; Wirth, Christophe; Hunte, Carola

doi:10.1016/bs.mie.2014.12.029

Cited by 4 publications

(3 citation statements)

References 43 publications

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“…To identify conformation-dependent and functional antibodies, antigen quality is critical. For antibody analyses, it is recommended to use a native ELISA format, such as indirect immobilization of the membrane protein via protein tags (Lim, Fang, & Williams, 2011;Mir et al, 2015). Direct binding of membrane protein antigen to the surfaces of ELISA plates is expected to distort the protein structure (Mir et al, 2015).…”

Section: Analyses Of the Resulting Antibodiesmentioning

confidence: 99%

Generation of High‐Specificity Antibodies against Membrane Proteins Using DNA‐Gold Micronanoplexes for Gene Gun Immunization

et al. 2018

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Membrane proteins are the molecular interface of the cell and its environs; however, studies of membrane proteins are highly technically challenging, mainly due to instability of the isolated protein. Towards the production of antibodies that recognize properly folded and stabilized forms of membrane protein antigen, we describe a DNA-based immunization method for mice that expresses the antigen in the membranes of dendritic cells, thus allowing direct presentation to the immune system. This genetic immunization approach employs a highly efficient method of biolistic delivery based on DNA-gold micronanoplexes, which are complexes of micron-sized gold particles that allow dermal penetration and nanometer-sized gold particles that provide a higher surface area for DNA binding than micron gold alone. In contrast to antibodies derived from immunizations with detergent-solubilized protein or with protein fragments, antibodies from genetic immunization are expected to have a high capacity for binding conformational epitopes and for modulating membrane protein activity. © 2018 by John Wiley & Sons, Inc.

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Section: Analyses Of the Resulting Antibodiesmentioning

confidence: 99%

Generation of High‐Specificity Antibodies against Membrane Proteins Using DNA‐Gold Micronanoplexes for Gene Gun Immunization

et al. 2018

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“…Antibodies such as conventional IgG, Fab-fragments (fragment antigen binding), scFv (single-chain variable fragments) and Camelid hcAb (heavy chain IgG) were used to reinforce protein crystallization, mainly for membrane protein Advanced Biocrystallogenesis DOI: http://dx.doi.org/10.5772/intechopen.97162 co-crystallization. Latterly, nanobodies replaced the use of antibodies because production of antibodies is limited and obtained yield tends to be conformationally heterogeneous with limited solubility [43,44]. On the contrary, nanobodies are extremely stable and soluble antibody single-domain fragments that stabilize unstructured proteins by binding to them, form crystal contacts and thus speed up crystal growth.…”

Section: Co-crystallization With Ligands and Crystallization Chaperonesmentioning

confidence: 99%

Advanced Biocrystallogenesis

Smatanová¹,

Havlickova²,

Kascakova³

et al. 2022

Crystallization and Applications

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Nowadays, X-ray crystallography is one of the most popular structural biology methods. Successful crystallization depends not only on the quality of the protein sample, precipitant composition, pH or other biophysical and biochemical parameters, but also largely on the use of crystallization technique. Some proteins are difficult to be crystallized using basic crystallization methods; therefore, several advanced methods for macromolecular crystallization have been developed. This chapter briefly reviews the most promising advanced crystallization techniques and strategies as one of the efficient tools for crystallization of macromolecules. Crystallization in capillaries, gels, microfluidic chips, electric and magnetic fields as well as crystallization under microgravity condition and crystallization in living cells are briefly described.

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“…Several studies evaluating the quantity and quality of Fv fragments produced from different expression systems have been performed. Bicistronic V L ‐V H overexpression in the Escherichia coli periplasm results in relatively low yields (0.5–1 mg per liter of culture) . Recovery of functional Fv fragments from E. coli cytoplasmic inclusion bodies requires a laborious and time‐consuming refolding process during purification .…”

Section: Introductionmentioning

confidence: 99%

The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment‐mediated crystallography

et al. 2016

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Fv antibody fragments have been used as co-crystallization partners in structural biology, particularly in membrane protein crystallography. However, there are inherent technical issues associated with the large-scale production of soluble, functional Fv fragments through conventional methods in various expression systems. To circumvent these problems, we developed a new method, in which a single synthetic polyprotein consisting of a variable light (V ) domain, an intervening removable affinity tag (iRAT), and a variable heavy (V ) domain is expressed by a Gram-positive bacterial secretion system. This method ensures stoichiometric expression of V and V from the monocistronic construct followed by proper folding and assembly of the two variable domains. The iRAT segment can be removed by a site-specific protease during the purification process to yield tag-free Fv fragments suitable for crystallization trials. In vitro refolding step is not required to obtain correctly folded Fv fragments. As a proof of concept, we tested the iRAT-based production of multiple Fv fragments, including a crystallization chaperone for a mammalian membrane protein as well as FDA-approved therapeutic antibodies. The resulting Fv fragments were functionally active and crystallized in complex with the target proteins. The iRAT system is a reliable, rapid and broadly applicable means of producing milligram quantities of Fv fragments for structural and biochemical studies.

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Generation of Recombinant Antibody Fragments for Membrane Protein Crystallization

Cited by 4 publications

References 43 publications

Generation of High‐Specificity Antibodies against Membrane Proteins Using DNA‐Gold Micronanoplexes for Gene Gun Immunization

Generation of High‐Specificity Antibodies against Membrane Proteins Using DNA‐Gold Micronanoplexes for Gene Gun Immunization

Advanced Biocrystallogenesis

The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment‐mediated crystallography

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