Synthesis of Single and Tandem β-Clamp Miniprotein Binder Constructs for Expression and Biochemical Validation

Funding Credits and Construct Naming Perk

This project will be funded through ResearchHub Funding Credits. This mechanism is well suited to the project because the funding goal is directly tied to a tangible product - the synthesis of two β-clamp miniprotein expression constructs.

To recognize the individual or individuals who make the work possible, this proposal includes a construct-naming opportunity. The two constructs to be named are:

the single-miniprotein β-clamp binder
the tandem double-miniprotein β-clamp binder

If one donor funds the full synthesis order, that donor may name both constructs. If two donors split the full support, each donor may name one construct. If the project is funded by a larger group of smaller contributors, the construct names will be selected by a donor vote.

In the group-funding scenario, each donor who contributes Funding Credits will be invited to submit proposed names for the constructs. After funding closes, eligible proposed names will be placed into a simple public vote using ResearchHub comments. Each donor will receive one vote per construct, regardless of contribution size, so that the naming process reflects community participation rather than only contribution amount. The top eligible name for each construct will become its official project name.

These donor-selected names will be used as the official construct names throughout the project, including in ResearchHub Lab Notebook updates, project summaries, figures, presentations, and data files where appropriate. If the work is successful and the results are developed into a scientific manuscript, the construct names may also appear in the eventual journal article, figure labels, supplementary information, or acknowledgments, subject to scientific and editorial standards.

The naming tradition is consistent with the long history of memorable names in biology, where distinctive gene, protein, and strain names have often helped scientists remember the story behind the experiment. Creative names are welcome. However, proposed names must be appropriate for scientific communication: short, non-offensive, non-trademarked, and not misleading about therapeutic efficacy, clinical status, disease cure, or regulatory approval.

1. Abstract

This nano-grant will fund synthesis of two pET28 expression constructs encoding a prioritized BindCraft-designed miniprotein binder to the mycobacterial β-clamp. The β-clamp is an essential DNA replication and repair hub, and our prior work established the clamp-binding groove as a tractable target for competitive inhibition. Preliminary computational work has already generated 90 filtered BindCraft miniproteins, from which we selected one compact, globular design predicted to overlap the native clamp-binding region used by DnaE1, ImuB, and related partners. The proposed work will synthesize two constructs: a single miniprotein and a tandem double miniprotein separated by a 20-amino-acid linker. The tandem construct tests whether bivalent display improves apparent affinity through avidity, increased local concentration, or slower complete dissociation. The project will be led by Scott Nelson at Iowa State University using existing bacterial expression, protein purification, β-clamp purification, fluorescence-polarization, and ITC infrastructure. The timeline is eight weeks from ordering to pilot biochemical testing. Construct status, expression results, purification data, and binding data will be shared through a public ResearchHub Lab Notebook, with raw data archived through Open Science Framework.

2. Introduction

Scientific rationale and target background

Tuberculosis remains a major global health problem. The World Health Organization reported 1.23 million TB deaths in 2024, including 150,000 deaths among people with HIV, and an estimated 10.7 million new TB cases worldwide. Multidrug-resistant TB remains a public health crisis and health-security threat, with only about two in five people with drug-resistant TB accessing treatment in 2024. [11]

The bacterial β-clamp is an essential DNA replication processivity factor and a protein-protein interaction hub. Sliding clamps encircle duplex DNA and tether DNA polymerases and other replication/repair proteins to the template. In bacteria, the β-clamp is a homodimer, and each monomer contains a peptide-binding pocket near the interdomain connector loop. Because many replication and repair proteins bind this surface through short clamp-binding motifs, the β-clamp groove is an attractive site for competitive inhibition. [1]

In mycobacteria, β-clamp partner recruitment is relevant not only to DNA replication but also to DNA damage tolerance and mutagenic repair. DnaE2, for example, contributes to survival after DNA damage and the emergence of drug resistance in Mycobacterium tuberculosis. [2] Our recent ResearchHub Journal article, Structural Basis for Differential Affinity and Competitive Binding of DNA Polymerase Peptides to the Mycobacterial β-Clamp, established a structural and biochemical framework for studying mycobacterial clamp-binding peptides, including binding and competition assays that can now be used to test designed miniproteins. [4]

Why miniproteins?

De novo designed miniproteins are now a credible modality for targeting defined protein surfaces. Cao et al. reported designed miniproteins that bound diverse targets with nanomolar to picomolar affinities. [3] RFdiffusion and related deep-learning methods have expanded the ability to design protein structures and protein binders from molecular specifications. [10] BindCraft extends this approach as an automated, open-source pipeline for de novo binder design using AlphaFold2-guided optimization and filtering; the published BindCraft study reported experimental success rates of 10–100% across tested targets, with some binders reaching nanomolar affinity without high-throughput experimental screening. [9]

The key conceptual move in this project is to advance beyond linear clamp-binding peptides. A folded miniprotein may present a larger, more preorganized binding surface than a short linear motif. This could improve affinity, specificity, and downstream engineering potential while still keeping the construct small enough for inexpensive synthesis and bacterial expression.

Why a tandem miniprotein?

The second construct fuses two copies of the same miniprotein with a 20-amino-acid linker. This is a deliberate test of whether bivalent display improves apparent binding. In multivalent systems, multiple ligands on one molecule can engage multiple receptors or binding sites, often producing a collective interaction stronger than the corresponding monovalent interaction. This is commonly described as avidity or functional affinity. [8]

For this project, a tandem miniprotein could improve apparent β-clamp binding in several ways. First, after one miniprotein copy binds, the second copy is tethered near the target rather than freely diffusing in solution. This raises the effective local concentration of the second binding unit and may favor intramolecular engagement or rapid rebinding after partial dissociation. Effective molarity and linker length are known to influence intramolecular protein-ligand binding. [8] Second, full dissociation of a bivalent construct generally requires both binding units to disengage. Even if one copy releases, the second can keep the molecule in a “sticky orbit” near the clamp long enough for rebinding, reducing the observed off-rate. Third, the β-clamp is dimeric, with partner-binding pockets on the clamp architecture, making it plausible that a properly linked tandem binder could engage two nearby clamp-binding surfaces or at least rebind efficiently to the same functional groove. [1]

There is strong precedent for this strategy. Linker length can strongly affect avidity in bivalent ligand-receptor systems. [6] Bivalent protein inhibitors have also shown increased potency relative to monovalent forms, including in soluble protein systems where avidity is explained by enhanced local concentration and rebinding. [8] BindCraft itself used a bivalent Fc-fusion format for PD-1 binder testing, where avidity and extremely slow dissociation complicated exact Kd determination for the strongest binder. [9]

Thus, the tandem construct is a low-cost, high-information experiment. If it binds more tightly than the single miniprotein, it immediately motivates linker optimization. If it does not, that result still clarifies that the next design cycle should prioritize single-domain affinity, alternative linker geometry, or a different binder orientation.

Why a ResearchHub nano-grant?

This project is a clean fit for a ResearchHub nano-grant because the requested funds support a small but decisive bottleneck: obtaining the physical DNA constructs. The computational design work has already been completed, and the downstream expression, purification, and β-clamp binding assays are available in my laboratory. The grant turns a prioritized in silico binder into two experimentally testable reagents.

The project also extends the open-science workflow used in my prior ResearchHub nano-grant on β-clamp peptide inhibitors. In that previous proposal, the workflow moved from computational screening to low-cost synthesis and biochemical validation. This proposal follows the same logic but moves the modality from linear peptides to folded miniproteins.

Hypotheses

H1. Primary hypothesis: the prioritized BindCraft miniprotein will express solubly in E. coli and can be purified through its N-terminal His-tag.

Primary outcome: detectable soluble expression of the single-miniprotein construct in E. coli and enrichment/purification of the encoded His-tagged miniprotein by immobilized metal affinity chromatography.

Operational criteria for success: after induction, the miniprotein should be visible at the expected molecular weight in the soluble lysate fraction by SDS-PAGE and should be enriched in the elution fraction after Ni-NTA or equivalent His-tag purification.

Quality-control milestone: receipt of a sequence-verified pET28 expression plasmid will be documented, but this is not the biological test of H1. The hypothesis is focused on soluble expression and purification.

Interpretation: successful soluble expression and His-tag purification would validate the selected BindCraft design as experimentally tractable and suitable for downstream β-clamp binding assays. Failure would indicate that the next design cycle should emphasize expression, solubility, scaffold selection, construct boundaries, linker/fusion strategy, or alternative purification tags.

H2. Primary hypothesis: the single miniprotein will bind or compete for the mycobacterial β-clamp binding groove.

Primary outcome: detectable β-clamp interaction by pull-down, fluorescence-polarization competition, ITC, or a related binding assay.

Interpretation: detectable binding would validate the selected BindCraft design as a β-clamp binder. Lack of binding, if expression is successful, would suggest that the predicted interface or site overlap is insufficient and should be redesigned.

H3. Secondary hypothesis: the tandem double-miniprotein construct will show stronger apparent β-clamp binding than the single-miniprotein construct.

Secondary outcome: lower apparent Kd, lower apparent Ki, stronger competition, stronger pull-down enrichment, or slower apparent dissociation for the double construct relative to the single construct.

Interpretation: improved binding would support an avidity/local-concentration mechanism and justify linker optimization. No improvement would suggest that the tandem geometry is unproductive, sterically hindered, or unable to access two binding events under the assay conditions.

3. Methods

Construct design and synthesis

The selected BindCraft miniprotein sequence will be submitted to Gene Universal for synthesis and cloning into pET28. Both constructs will be codon optimized for bacterial expression and designed with an N-terminal His-tag.

Construct 1: Single miniprotein

Feature	Specification
Vector	pET28
Insert	One prioritized BindCraft-designed β-clamp-binding miniprotein
Optimization	E. coli codon optimized
Tag	N-terminal His-tag
Vendor deliverable	Sequence-verified plasmid DNA, plasmid map, Sanger files, COA

Construct 2: Double miniprotein

Feature	Specification
Vector	pET28
Insert	Miniprotein copy 1 + 20-aa linker + miniprotein copy 2
Optimization	E. coli codon optimized
Tag	N-terminal His-tag
Vendor deliverable	Sequence-verified plasmid DNA, plasmid map, Sanger files, COA

The 20-amino-acid linker will be (GGGGS) X 4. Twist Biosciences was selected because it is the lowest-cost vendor I have found for this order. Their deliverables are purified plasmid DNA, electronic plasmid maps, Sanger sequencing files, and COA. [4,5]

Plasmid receipt and expression screening

Upon receipt, the plasmids will be transformed into our standard E. coli expression strain, BL21(DE3). Small-scale expression tests will compare induced and uninduced cultures, as well as soluble and insoluble fractions, by SDS-PAGE. Because both constructs carry an N-terminal His-tag, initial purification will use standard immobilized metal affinity chromatography.

Key experimental readouts will include:

Readout	Purpose
Vendor plasmid map and Sanger verification	Confirms construct identity
SDS-PAGE expression screen	Determines whether each construct expresses
Soluble/insoluble fraction comparison	Determines whether each construct is soluble
Small-scale His-tag purification	Determines whether sufficient material can be obtained for pilot binding assays
Comparison of single vs. tandem behavior	Identifies whether tandem fusion affects expression, solubility, degradation, or purification

Pilot biochemical validation

If soluble protein is obtained, the purified miniproteins will be tested against purified mycobacterial β-clamp using assays already available in the lab. Depending on protein yield and behavior, initial tests will use one or more of the following:

Assay/readout	Purpose
Pull-down or co-purification assay	Rapid qualitative test for β-clamp association
Fluorescence-polarization competition	Tests displacement of a fluorescent clamp-binding peptide tracer
ITC	Measures direct binding if sufficient protein is available
SDS-PAGE/SEC behavior	Checks purity, degradation, and aggregation
Single vs. tandem comparison	Tests whether tandem architecture improves apparent affinity or competition

The single and tandem constructs will be compared directly whenever possible. If the tandem construct shows a stronger apparent signal, follow-up work will focus on linker length, linker composition, and binder orientation. If the tandem construct performs worse, the result will be interpreted as evidence that the linker geometry or tandem architecture is not yet productive.

Team, facilities, and timeline

Role	Investigator	Position	Expertise
PI	Scott Nelson, PhD	Professor, Iowa State University, Ames, IA	Biochemistry and biophysics of DNA replication enzymes; β-clamp purification; peptide/protein binding assays; fluorescence polarization; ITC; protein expression and purification

Relevant facilities include standard bacterial expression and purification infrastructure, the ISU Protein Facility, and established access to β-clamp biochemical assays.

Estimated timeline

Phase	Timeline	Milestone
Construct finalization and ordering	Weeks 0–1	Submit two sequences to Gene Universal
Vendor synthesis and cloning	Weeks 1–3	Receive sequence-verified plasmids
Transformation and expression testing	Weeks 3–5	Determine expression and solubility
Small-scale purification	Weeks 5–6	Obtain His-tagged miniprotein material
Pilot binding/competition assays	Weeks 6–8	Compare single vs. tandem constructs
Data posting and archiving	Ongoing, with final update by Week 8	ResearchHub notebook and DataShare deposition after IP review

Planned Analyses

Expression and purification analysis

Expression will be scored by SDS-PAGE comparison of induced versus uninduced cultures. Solubility will be assessed by comparing soluble and insoluble fractions. Purification success will be evaluated by SDS-PAGE band intensity, apparent purity, and approximate yield after His-tag purification.

Result	Interpretation
Single construct expresses and purifies	Design is experimentally tractable
Single construct fails to express	Redesign for solubility/expression is needed before binding conclusions can be drawn
Tandem construct expresses less well than single	Tandem fusion may affect folding, stability, or solubility
Tandem construct expresses comparably to single	Tandem format is suitable for direct binding comparison

Binding and competition analysis

For fluorescence-polarization competition experiments, raw polarization values will be background-corrected and normalized to the no-competitor and maximum-competition controls. Competition curves will be fit to estimate apparent Ki or IC50 values when the data quality supports quantitative fitting. If the tandem construct produces a curve that cannot be described by a simple monovalent model, the result will be reported as an apparent competition value rather than overinterpreted as a simple thermodynamic Kd.

For ITC, raw heat traces will be integrated, baseline-corrected, and fit initially using a simple binding model. If the tandem construct displays complex behavior inconsistent with a one-site model, the result will be reported cautiously as apparent binding behavior, with stoichiometry and model limitations stated explicitly.

For pull-down or co-purification experiments, enrichment will be interpreted qualitatively or semi-quantitatively using band intensity. A positive tandem result will require stronger β-clamp association than the single construct under the same conditions.

Hypothesis-specific predictions

Hypothesis	Prediction	Interpretation if supported	Interpretation if not supported
H1	Single construct expresses and purifies as a soluble His-tagged protein	BindCraft design is experimentally tractable	Redesign should prioritize expression/solubility
H2	Single construct binds or competes for β-clamp	Selected design is a valid β-clamp binder	Interface redesign or alternate candidate selection is needed
H3	Tandem construct shows stronger apparent binding than single	Avidity/local concentration is productive	Tandem geometry, linker, or orientation is not productive

Data Management

The open-science plan has two layers. First, project progress will be documented in a public ResearchHub Lab Notebook, including ordering status, construct descriptions, expression screens, purification results, assay outcomes, and interpretation. ResearchHub Notebook supports drafting and publishing research content directly in the ResearchHub web application. [14] Second, raw and processed data will be archived through Open Science Framework.

4. Pilot Data: Preliminary Work and BindCraft Design

This nano-grant is not requesting funds to begin miniprotein design from scratch. The computational design stage has already been completed.

We used BindCraft to generate candidate miniprotein binders against the mycobacterial β-clamp surface. BindCraft is well suited for this task because it generates de novo protein binders using AlphaFold2-guided design, sequence optimization, and structural/interface filters. The published BindCraft study describes it as an automated open-source pipeline for de novo protein-binder design and reports experimental success rates of 10–100% across tested targets. [9]

For this project, the design goal was not simply to generate any β-clamp-binding protein. The goal was to generate small, expressible miniproteins that engage the same functional binding groove used by native clamp partners, including DnaE1, ImuB, and related β-clamp-interacting proteins. This matters because the therapeutic objective is to disrupt biologically relevant clamp-partner recruitment, not merely to bind an arbitrary β-clamp surface.

The BindCraft campaign produced 90 miniprotein designs that passed the computational filters. These filtered designs were ranked and manually inspected. The ranking considered both numerical design metrics and practical experimental features:

Selection criterion	Rationale
Predicted binder fold quality	Increases likelihood of soluble, folded recombinant expression
Compactness and globularity	Favors a true miniprotein rather than an extended peptide-like structure
Predicted interface quality	Prioritizes designs likely to maintain target engagement
Overlap with DnaE1/ImuB binding region	Focuses on biologically relevant clamp-partner competition
Suitability for bacterial expression	Enables a fast pET28/His-tag workflow
Suitability for tandem fusion	Enables testing of bivalent architecture using a 20-aa linker

From the 90 filtered designs, we selected one candidate that combines two features that are especially important for this first synthesis experiment. First, the design is a small globular fold, making it a strong candidate for soluble recombinant expression in E. coli. Second, the predicted binding interface shows strong spatial overlap with the β-clamp binding site occupied by native peptide motifs from DnaE1, ImuB, and related clamp-interacting proteins. In other words, the chosen design is not just predicted to bind the β-clamp; it is predicted to bind at the functionally relevant site where competition with native replication and repair proteins should be possible. The chosen designs are shown below.

Figure 1 (above). Single miniprotein model bound to one β-clamp subunit.
Predicted structure of the selected BindCraft miniprotein binder positioned at the β-clamp partner-binding region. The miniprotein is shown in blue against one β-clamp subunit, highlighting its compact globular fold and overlap with the functional clamp-binding site targeted by native partners such as DnaE1 and ImuB.

Figure 2 (above). Tandem miniprotein model bound to the β-clamp dimer.
Predicted placement of the double-miniprotein construct on the β-clamp dimer surface. The two miniprotein copies are shown in yellow and blue, with the 20-amino-acid linker shown in red. This model illustrates the rationale for testing a tandem construct: after one miniprotein binds, the second copy may be held near the second clamp-binding groove, potentially increasing apparent affinity through avidity or rapid rebinding.

This computational triage substantially de-risks the proposed synthesis. The requested funds will convert a prioritized BindCraft hit into two experimentally testable expression constructs: a single-copy miniprotein and a tandem two-copy miniprotein with a 20-amino-acid linker.

These pilot data are distinct from the main study. The pilot work consists of computational design, filtering, ranking, and manual selection. The main study funded here is the synthesis of the two constructs and their initial expression and biochemical validation.

5. Budget

Total requested: $245

Item	Cost
Single miniprotein construct in pET28, E. coli codon optimized, N-terminal His-tag, sequence verified	$100
Double miniprotein construct in pET28, 20-aa linker, E. coli codon optimized, N-terminal His-tag, sequence verified	$120
Shipping and handling	$25
Total requested	$245

Budget justification. The request is limited to outsourced synthesis and cloning of the two expression constructs.

6. References

Altieri, A. S. & Kelman, Z. DNA sliding clamps as therapeutic targets. Front. Mol. Biosci. 5, 87 (2018).
Boshoff, H. I. M., Reed, M. B., Barry, C. E. III & Mizrahi, V. DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113, 183–193 (2003).
Cao, L. et al. Design of protein-binding proteins from the target structure alone. Nature 605, 551–560 (2022).
Kapur, M., Gray, O. J., Sen, S., Honzatko, R. B. & Nelson, S. W. Structural Basis for Differential Affinity and Competitive Binding of DNA Polymerase Peptides to the Mycobacterial β-Clamp. ResearchHub Journal doi:10.55277/rhj.cqzht3xe.5 (2025).
Krishnamurthy, V. M., Semetey, V., Bracher, P. J., Shen, N. & Whitesides, G. M. Dependence of effective molarity on linker length for an intramolecular protein-ligand system. J. Am. Chem. Soc. 129, 1312–1320 (2007).
Lacham-Hartman, S. et al. Avidity observed between a bivalent inhibitor and an enzyme monomer with a single active site. PLoS ONE 16, e0249616 (2021).
Mack, E. T., Snyder, P. W., Perez-Castillejos, R., Bilgiçer, B., Moustakas, D. T., Butte, M. J. & Whitesides, G. M. Dependence of avidity on linker length for a bivalent ligand-bivalent receptor model system. J. Am. Chem. Soc. 134, 333–345 (2012).
Mammen, M., Choi, S.-K. & Whitesides, G. M. Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Int. Ed. 37, 2754–2794 (1998).
Pacesa, M. et al. One-shot design of functional protein binders with BindCraft. Nature 646, 483–492 (2025).
Watson, J. L. et al. De novo design of protein structure and function with RFdiffusion. Nature 620, 1089–1100 (2023).
World Health Organization. Tuberculosis fact sheet. Updated 24 March 2026.

Synthesis of Single and Tandem β-Clamp Miniprotein Binder Constructs for Expression and Biochemical Validation

Synthesis of Single and Tandem β-Clamp Miniprotein Binder Constructs for Expression and Biochemical Validation

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