Constructing and purifying megabodies starting from individual nanobody sequences
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.
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Due to technical limitations, Tables 1-2 can be found in the Supplementary Files section.
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Table 1. PCR components and setting.
Table 1. PCR components and setting.
Posted 07 Jan, 2021
Constructing and purifying megabodies starting from individual nanobody sequences
Posted 07 Jan, 2021
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.
© Research Square 2021