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Engineering a Minimal Leucine-rich Repeat IgG-binding Module

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Abstract

Sea lamprey immunization can yield leucine-rich repeat (LRR) protein binders analogous to globular antibodies developed from mammals. A novel minimal LRR was discovered through lamprey immunization with human immunoglobulin G Fc domain (IgG Fc). Initial attempts to solubly express this LRR protein, VLRB.IgGFc, in Escherichia coli proved challenging, so it was analyzed using the cell-free method ribosome display. In ribosome display, VLRB.IgGFc was found to bind specifically to the Fc domain of IgG, with little observed cross-reactivity to IgA or IgM. The minimal repeat protein architecture of VLRB.IgGFc may facilitate modular LRR extensions to incorporate additional or augmented functionality within a continuous, structurally defined scaffold. We exploited this modularity to design a chimera of a well-characterized, soluble LRR repebody and the initially insoluble VLRB.IgGFc to produce soluble Repe-VLRB.IgGFc. The minimal IgG Fc–binding module, Repe-VLRB.IgGFc, and future-engineered variants thereof should be useful additions to the biotechnological toolbox for detecting, purifying, or targeting IgGs. More generally, this two-step approach of minimal LRR binder discovery via sea lamprey immunization followed by modular augmentation of functionality may be of general utility in protein engineering.

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Acknowledgements

We thank Christina Larson, Sally Mayasich, and Ben Clarke for their support in sea lamprey husbandry and handling.

Funding

This work was supported by a 3M Science and Technology Fellowship (G.C.M) and by the University of Minnesota (C.A.S.).

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Authors and Affiliations

  1. Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN, 55455, USA

    George C. Markou

  2. Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, 55455, USA

    Ayako Ohoka & Casim A. Sarkar

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  1. George C. Markou
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  2. Ayako Ohoka
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  3. Casim A. Sarkar
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Contributions

G.C.M. and C.A.S. conceived the idea; G.C.M. and A.O. carried out the experiments and analyses; G.C.M. and C.A.S. wrote the manuscript.

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Correspondence to Casim A. Sarkar.

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Markou, G.C., Ohoka, A. & Sarkar, C.A. Engineering a Minimal Leucine-rich Repeat IgG-binding Module. Appl Biochem Biotechnol 194, 1636–1644 (2022). https://doi.org/10.1007/s12010-021-03768-6

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  • DOI: https://doi.org/10.1007/s12010-021-03768-6

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