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Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

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Published on: November 19, 2020

CDR1 Composition Can Affect Nanobody Recombinant Expression Yields.

Marco Orlando¹, Sara Fortuna², Sandra Oloketuyi³

¹Department of Biotechnology and Life Sciences, University of Insubria, Via J. H. Dunant 3, 21100 Varese, Italy.

|September 28, 2021

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Researchers optimized nanobodies by modifying CDR1, enhancing yields but slightly reducing affinity. This protein engineering strategy offers a novel approach for improving antibody characteristics.

Area of Science:

Biochemistry
Protein Engineering
Immunology

Background:

Nanobody discovery via biopanning is efficient but often yields proteins needing biophysical optimization.
Complementarity-determining regions (CDRs) are typically conserved due to their role in antigen binding.
Specific CDRs can present instability or suboptimal characteristics requiring targeted engineering.

Purpose of the Study:

To develop and evaluate a mutagenesis strategy targeting CDR1 for improved nanobody biophysical properties.
To investigate the impact of loop modification on nanobody yield and antigen-binding affinity.
To elucidate the structural and dynamic effects of engineering interventions in CDR1.

Main Methods:

Utilized the PROSS program to identify CDR1 as an instability hot spot.

Keywords:

in silico modeling nanobody CDRs nanobody engineering rational mutagenesis

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Employed a mutagenesis strategy involving the insertion of an extra glycine residue into CDR1.

Performed accurate modeling and atomistic molecular dynamics simulations to analyze structural changes.

Main Results:

The glycine insertion strategy significantly increased nanobody yields.
A moderate loss of antigen-binding affinity was observed as a trade-off for increased yield.
Molecular dynamics simulations provided detailed insights into the effects of the glycine insertion on loop flexibility and interactions.

Conclusions:

Targeted modification of CDR1, specifically by altering loop flexibility, can enhance nanobody production yields.
Engineering CDRs presents a viable strategy for optimizing nanobody characteristics, balancing yield and affinity.
Computational modeling is crucial for understanding the mechanistic basis of protein engineering outcomes in nanobodies.