# Selection system for identifying protein-specific folding tags that enable purification of native cytokines from E. coli

> **NIH NIH R43** · POTOMAC AFFINITY PROTEINS, LLC · 2021 · $224,806

## Abstract

Project Summary: Our overall objective is to develop and test a selection system for evolving tags that
enable folding of cytokines in E. coli and facile purification of the tag-free, native protein. Selection is
based on the discovery that unfolded proteins restrict growth of the E. coli host cell when co-expressed
with an engineered restriction protease. A tag that facilitates the folding of an otherwise poorly-folded
protein rescues growth. Using the selection method, we will develop tags enabling purification of key
cytokines needed to create defined, synthetic cell culture media. Our approach employs random
mutagenesis of a tandem tag of Protein G domains (GA-GB) and selection using a restriction protease
system that we will develop. We will evolve folding tags for four targets and expand the envelop of
recombinant proteins that can be purified in E coli. Specific Aims are: Construct plasmids for co-
expression of GA-GB-target proteins with the restriction protease; Construct random libraries of GA-GB-
target protein; Select for escape mutants; Analyze of mutations that allow escape; Purify proteins using
newly-evolved tags; Test physical and biological properties of purified proteins. Feasibility will be
determined by whether we can evolve effective folding tags for all four targets. An effective folding tag is
defined as one that allows purification of ≥50mg of native, biologically-active cytokine per L of culture. At
the end of Phase I we will have developed methods for identifying effective folding tags for two major
structural classes of cytokines. We will also have learned whether evolved folding tags for a structural
class of cytokine are similar. This knowledge will simplify identification of folding tags for new cytokine
targets as well as other high-value proteins. In Phase II we would evolve tags for 17 major cytokines
used in cell culture.

## Key facts

- **NIH application ID:** 10256900
- **Project number:** 1R43GM140633-01A1
- **Recipient organization:** POTOMAC AFFINITY PROTEINS, LLC
- **Principal Investigator:** PHILIP N BRYAN
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $224,806
- **Award type:** 1
- **Project period:** 2021-05-01 → 2022-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10256900

## Citation

> US National Institutes of Health, RePORTER application 10256900, Selection system for identifying protein-specific folding tags that enable purification of native cytokines from E. coli (1R43GM140633-01A1). Retrieved via AI Analytics 2026-06-25 from https://api.ai-analytics.org/grant/nih/10256900. Licensed CC0.

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