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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
biochemistry, genetics and molecular biology
Combining in-situ proteolysis and microseed matrix screening to promote crystallization of PrP
c
-nanobody complexes
Protein Engineering, Design and Selection, Volume 24, No. 9, Year 2011
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Description
Prion proteins (PrPs) are difficult to crystallize, probably due to their inherent flexibility. Several PrPs structures have been solved by nuclear magnetic resonance (NMR) techniques; however, only three structures were solved by X-ray crystallography. Here we combined in-situ proteolysis with automated microseed matrix screening (MMS) to crystallize two different PrP C-nanobody (Nb) complexes. Nanobodies are single-domain antibodies derived from heavy-chain-only antibodies of camelids. Initial crystallization screening conditions using in-situ proteolysis of mouse prion (23-230) in complex with a nanobody (Nb-PrP-01) gave thin needle aggregates, which were of poor diffraction quality. Next, we used these microcrystals as nucleants for automated MMS. Good-quality crystals were obtained from mouse PrP (89-230)/Nb-PrP-01, belonged to the monoclinic space group P 1 21 1, with unit-cell parameters a = 59.13, b = 63.80, c = 69.79 Å, β = 101.96° and diffracted to 2.1Å resolution using synchrotron radiation. Human PrP (90-231)/Nb-PrP-01 crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 131.86, b = 45.78, c = 45.09 Å, β = 96.23° and diffracted to 1.5 Å resolution. This combined strategy benefits from the power of the MMS technique without suffering from the drawbacks of the in-situ proteolysis. It proved to be a successful strategy to crystallize PrP-nanobodies complexes and could be exploited for the crystallization of other difficult antigen-antibody complexes. © The Author 2011. Published by Oxford University Press. All rights reserved.
Authors & Co-Authors
Abskharon, Romany N.N.
Belgium, Brussels
Free Universities of Brussels
Belgium, Ghent
Vlaams Instituut Voor Biotechnologie
Soror, Sameh Hamdy
Belgium, Brussels
Free Universities of Brussels
Belgium, Ghent
Vlaams Instituut Voor Biotechnologie
Egypt, Helwan
Faculty of Pharmacy
Pardon, Els
Belgium, Brussels
Free Universities of Brussels
Belgium, Ghent
Vlaams Instituut Voor Biotechnologie
El Hassan, Hassan
Belgium, Brussels
Free Universities of Brussels
Belgium, Ghent
Vlaams Instituut Voor Biotechnologie
Legname, Giuseppe
Italy, Trieste
Scuola Internazionale Superiore Di Studi Avanzati
Steyaert, Jan
Belgium, Brussels
Free Universities of Brussels
Belgium, Ghent
Vlaams Instituut Voor Biotechnologie
Wohlkönig, Alexandre
Belgium, Brussels
Free Universities of Brussels
Belgium, Ghent
Vlaams Instituut Voor Biotechnologie
Statistics
Citations: 18
Authors: 7
Affiliations: 4
Identifiers
Doi:
10.1093/protein/gzr017
ISSN:
17410126
e-ISSN:
17410134
Research Areas
Cancer