# Computational de novo design of a disulfide-rich miniprotein synthetic library and its application to engineer binders to neutralizing epitopes on Clostridium difficile toxins TcdA and TcdB

> **NIH NIH R21** · INSTITUTE FOR PROTEIN INNOVATION, INC. · 2021 · $246,300

## Abstract

Project Summary
Disulfide-rich miniproteins are a powerful yet underutilized protein family for reagent, diagnostic and therapeutic
applications. They are hyperstable like small molecules, yet are large enough to bind specifically to protein
targets with high affinity and thus can be readily used to inhibit protein-protein interactions. Disulfide-rich
miniproteins occur naturally, but the natural molecules are challenging to engineer. There is a pressing need for
new technologies which enable disulfide-rich miniproteins to be engineered to bind to arbitrary protein targets,
as is routine for other protein scaffolds like antibodies. Here, we propose a computational de novo design
strategy using Rosetta to build a synthetic miniprotein library that will be generally useful for screening protein
affinity reagents.
The typical approach to creating protein libraries is to generate a large amount of random sequence diversity in
a localized area of a single protein structure. Most of these sequences will be unstable or unable to bind any
target (e.g. too polar or too nonpolar). Rather than use random sequences, we propose to explicitly design
each member of a synthetic disulfide-rich miniprotein library. Our design library will contain 106 different
miniproteins that display the widest possible variety of binding surfaces. The genes encoding this library will be
synthesized using oligo pools. To perform computational de novo design at this scale, we extended the
SEWING algorithm in Rosetta to generate hundreds of thousands of unique miniprotein structures and
sequences. We developed novel filters to assess design model quality and to quantify and compare protein
structures and surfaces.
As a test case of this approach, we will screen our disulfide-rich miniprotein library via yeast display for binders
to Clostridium difficile enterotoxins TcdA and TcdB. C. difficile is the leading cause of healthcare-related
infections in the USA, and these two proteins mediate its pathogenicity. At present, the only available
treatments for C. difficile enteric infection that target these toxin proteins are antibodies, which must be injected
and have poor efficacy. A hyperstable miniprotein binder to the same neutralizing epitope could be
administered orally. Therefore, this work presents a new frontier in de novo miniprotein engineering and library
design. It will also result in a source of novel, therapeutically interesting molecules for the treatment of C.
difficile infection.

## Key facts

- **NIH application ID:** 10110455
- **Project number:** 1R21AI156570-01
- **Recipient organization:** INSTITUTE FOR PROTEIN INNOVATION, INC.
- **Principal Investigator:** Christopher David Bahl
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $246,300
- **Award type:** 1
- **Project period:** 2020-12-10 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10110455

## Citation

> US National Institutes of Health, RePORTER application 10110455, Computational de novo design of a disulfide-rich miniprotein synthetic library and its application to engineer binders to neutralizing epitopes on Clostridium difficile toxins TcdA and TcdB (1R21AI156570-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10110455. Licensed CC0.

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