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Method Article
Constructing and purifying megabodies starting from individual nanobody sequences
Jan Steyaert
1: Structural Biology Brussels, Vrije Universiteit Brussel, VUB, Brussels, Belgium. 2: VIB-VUB Center for Structural Biology, VIB, Brussels, Belgium.
Corresponding Author
ORCiD: https://orcid.org/0000-0002-3825-874X
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Megabodies are engineered nanobodies that help overcome two major obstacles that limit the resolution of single-particle cryo-EM reconstructions: particle size and preferential orientation at the water-air interfaces. Here we describe how nanobodies can be rigidly grafted onto selected protein scaffolds (HopQ or YgjK) to increase their molecular weight while retaining the full antigen binding specificity and affinity. We also describe the protocols to purify these chimeric molecules from the periplasm of E. coli.
Keywords
megabody, nanobody, single-particle cryo-EM
Figure 1
Figure 2
Figure 3
Figure 4
Due to technical limitations, Tables 1-2 can be found in the Supplementary Files section.
This is a list of supplementary files associated with this preprint. Click to download.
- Table1.pdf
Table 1. PCR components and setting.
- Table1.pdf
Table 1. PCR components and setting.
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Associated Publications
Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM
by Tomasz Uchański, Simonas Masiulis, Baptiste Fischer, +15
Nature Methods (06 January, 2021)
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This is a preprint. It has not completed peer review.
Method Article
Constructing and purifying megabodies starting from individual nanobody sequences
Jan Steyaert
1: Structural Biology Brussels, Vrije Universiteit Brussel, VUB, Brussels, Belgium. 2: VIB-VUB Center for Structural Biology, VIB, Brussels, Belgium.
Corresponding Author
ORCiD: https://orcid.org/0000-0002-3825-874X
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 07 Jan, 2021
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Structural biology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Megabodies are engineered nanobodies that help overcome two major obstacles that limit the resolution of single-particle cryo-EM reconstructions: particle size and preferential orientation at the water-air interfaces. Here we describe how nanobodies can be rigidly grafted onto selected protein scaffolds (HopQ or YgjK) to increase their molecular weight while retaining the full antigen binding specificity and affinity. We also describe the protocols to purify these chimeric molecules from the periplasm of E. coli.
Figure 1
Figure 2
Figure 3
Figure 4
Due to technical limitations, Tables 1-2 can be found in the Supplementary Files section.
Associated Publications
Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM
by Tomasz Uchański, Simonas Masiulis, Baptiste Fischer, +15
Nature Methods (06 January, 2021)