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Title: Protein crystallization with microseed matrix screening: application to human germline antibody Fabs

Abstract

The power of microseed matrix screening is demonstrated in the crystallization of a panel of antibody Fab fragments. The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization.

Authors:
; ; ; ;  [1]
  1. Janssen Research and Development LLC, 1400 McKean Road, Spring House, PA 19477 (United States)
Publication Date:
OSTI Identifier:
22375697
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta crystallographica. Section F, Structural biology communications; Journal Volume: 70; Journal Issue: Pt 8; Other Information: PMCID: PMC4118815; PMID: 25084393; PUBLISHER-ID: nj5193; OAI: oai:pubmedcentral.nih.gov:4118815; Copyright (c) Obmolova et al. 2014; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CHAINS; CRYSTALLIZATION; CRYSTALS; DIFFRACTION; OPTIMIZATION; PROTEINS; SCREENING; VISIBLE RADIATION

Citation Formats

Obmolova, Galina, E-mail: gobmolov@its.jnj.com, Malia, Thomas J., Teplyakov, Alexey, Sweet, Raymond W., and Gilliland, Gary L., E-mail: gobmolov@its.jnj.com. Protein crystallization with microseed matrix screening: application to human germline antibody Fabs. United States: N. p., 2014. Web. doi:10.1107/S2053230X14012552.
Obmolova, Galina, E-mail: gobmolov@its.jnj.com, Malia, Thomas J., Teplyakov, Alexey, Sweet, Raymond W., & Gilliland, Gary L., E-mail: gobmolov@its.jnj.com. Protein crystallization with microseed matrix screening: application to human germline antibody Fabs. United States. doi:10.1107/S2053230X14012552.
Obmolova, Galina, E-mail: gobmolov@its.jnj.com, Malia, Thomas J., Teplyakov, Alexey, Sweet, Raymond W., and Gilliland, Gary L., E-mail: gobmolov@its.jnj.com. Wed . "Protein crystallization with microseed matrix screening: application to human germline antibody Fabs". United States. doi:10.1107/S2053230X14012552.
@article{osti_22375697,
title = {Protein crystallization with microseed matrix screening: application to human germline antibody Fabs},
author = {Obmolova, Galina, E-mail: gobmolov@its.jnj.com and Malia, Thomas J. and Teplyakov, Alexey and Sweet, Raymond W. and Gilliland, Gary L., E-mail: gobmolov@its.jnj.com},
abstractNote = {The power of microseed matrix screening is demonstrated in the crystallization of a panel of antibody Fab fragments. The crystallization of 16 human antibody Fab fragments constructed from all pairs of four different heavy chains and four different light chains was enabled by employing microseed matrix screening (MMS). In initial screening, diffraction-quality crystals were obtained for only three Fabs, while many Fabs produced hits that required optimization. Application of MMS, using the initial screens and/or refinement screens, resulted in diffraction-quality crystals of these Fabs. Five Fabs that failed to give hits in the initial screen were crystallized by cross-seeding MMS followed by MMS optimization. The crystallization protocols and strategies that resulted in structure determination of all 16 Fabs are presented. These results illustrate the power of MMS and provide a basis for developing future strategies for macromolecular crystallization.},
doi = {10.1107/S2053230X14012552},
journal = {Acta crystallographica. Section F, Structural biology communications},
number = Pt 8,
volume = 70,
place = {United States},
year = {Wed Jul 23 00:00:00 EDT 2014},
month = {Wed Jul 23 00:00:00 EDT 2014}
}
  • It is shown how protein crystallization results can be used to identify buffers that improve protein solubility and, in turn, crystallization success. An optimal solubility screen is described that uses the results of crystallization trials to identify buffers that improve protein solubility and, in turn, crystallization success. This screen is useful not only for standard crystallization experiments, but also can easily be implemented into any high-throughput structure-determination pipeline. As a proof of principle, the predicted novel-fold protein AF2059 from Archaeoglobus fulgidus, which was known to precipitate in most buffers and particularly during concentration experiments, was selected. Using the crystallization resultsmore » of 192 independent crystallization trials, it was possible to identify a buffer containing 100 mM CHES pH 9.25 that significantly improves its solubility. After transferring AF2059 into this ‘optimum-solubility’ buffer, the protein was rescreened for crystal formation against these same 192 conditions. Instead of extensive precipitation, as observed initially, it was found that 24 separate conditions produced crystals and the exchange of AF2059 into CHES buffer significantly improved crystallization success. Fine-screen optimization of these conditions led to the production of a crystal suitable for high-resolution (2.2 Å) structure determination.« less
  • Parathyroid hormone-related protein (PTHrP) plays an important role in regulating embryonic skeletal development and is abnormally regulated in the pathogenesis of skeletal complications observed with many cancers and osteoporosis. It exerts its action through binding to a G-protein-coupled seven-transmembrane cell-surface receptor (GPCR). Structurally, GPCRs are very difficult to study by X-ray crystallography. In this study, a monoclonal antibody Fab fragment which recognizes the same region of PTHrP as its receptor, PTH1R, was used to aid in the crystallization of PTHrP. The resultant protein complex was crystallized using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitant. The crystals belongedmore » to the orthorhombic space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 72.6, b = 96.3, c = 88.5 {angstrom}, and diffracted to 2.0 {angstrom} resolution using synchrotron radiation. The crystal structure will shed light on the nature of the key residues of PTHrP that interact with the antibody and will provide insights into how the antibody is able to discriminate between PTHrP and the related molecule parathyroid homone.« less
  • Antigen-free Fab fragment of mAbR310, which recognizes (R)-HNE modified protein, has been crystallized. Initial phases have been obtained by molecular replacement. 4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE–histidine Michael addition-type adduct possessing three chiral centres in the cyclic hemiacetal structure. Monoclonal antibodies against HNE-modified protein have been widely used for assessing oxidative stress in vitro and in vivo. Here, the purification, crystallization and preliminary crystallographic analysis of a Fab fragment of novel monoclonal antibody R310 (mAbR310), which recognizes (R)-HNE-modified protein, are reported. The Fab fragment of mAbR310 was obtained bymore » digestion with papain, purified and crystallized. Using hanging-drop vapour-diffusion crystallization techniques, crystals of mAbR310 Fab were obtained. The crystal belongs to the monoclinic space group C2 (unit-cell parameters a = 127.04, b = 65.31, c = 64.29 Å, β = 118.88°) and diffracted X-rays to a resolution of 1.84 Å. The asymmetric unit contains one molecule of mAbR310, with a corresponding crystal volume per protein weight of 2.51 Å{sup 3} Da{sup −1} and a solvent content of 51.0%.« less
  • No abstract prepared.