# Fast, flexible, and economical system for affinity-tag protein purification and tag removal

> **NIH NIH R44** · POTOMAC AFFINITY PROTEINS, LLC · 2020 · $729,996

## Abstract

Project Summary: Our long-term objective is to commercialize a fast, simple, flexible, and economical
system for affinity-tag protein purification and tag removal. This technology was developed as a result of
NIH-funded studies on the folding, stability and enzymology of the Bacillus protease subtilisin. The
transformative purification technology combines three components: 1) a regulated, highly-specific
protease (Psub) that is constitutively inactive; 2) a small molecule (imidazole) that activates the Psub; 3)
a high-affinity inhibitor (Pro). Immobilized Psub (in the off-state) can capture a Pro-fusion protein from a
cellular extract, allowing contaminants to be washed away. Subsequent addition of imidazole to the
immobilized complex releases pure, tag-free target protein and leaves the Pro-tag tightly bound to Psub.
Recombinant proteins are frequently fused with other proteins or peptides to facilitate expression and
purification. The tags enable target protein binding in affinity purification, but ultimately must be
processed by a site-specific protease. Tag removal, however, is frequently expensive, inefficient, and
sometimes problematic. The technical innovation of our system is the integration of seamless tag
removal into the purification process. This provides simplicity, efficiency, and robustness that is not
available in any other system. Improved understanding of protein engineering principles has resulted in
highly-efficient Psubs and Pro affinity tags. The experimental plan has two goals. The first is to generate
economical materials for purification of standard proteins without tags. The second goal more ambitious
goal is to develop methods for purifying high-value proteins that are either difficult or impossible with
current methods. The Specific Aims are: 1) Evaluation of site-specific immobilization methods for Psub;
2) Cloning of challenging, high-value target proteins; 3) Expression-extraction-purification of high-value
target proteins; 4) Optimization of the purification of challenging proteins; 5) Testing physical and
biological properties of purified proteins. Better purification technology should advance in all areas of
biology but have a particular impact on the production of growth factors needed for the growth, survival,
and differentiation of therapeutic cells.

## Key facts

- **NIH application ID:** 9969583
- **Project number:** 5R44GM126676-03
- **Recipient organization:** POTOMAC AFFINITY PROTEINS, LLC
- **Principal Investigator:** PHILIP N BRYAN
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $729,996
- **Award type:** 5
- **Project period:** 2019-07-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9969583

## Citation

> US National Institutes of Health, RePORTER application 9969583, Fast, flexible, and economical system for affinity-tag protein purification and tag removal (5R44GM126676-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9969583. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*