Proximity-Based Sortase Protein Conjugation

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Solution Overview

Problem

Current methods for site-specific protein conjugation, such as those using unnatural amino acids, enzyme-mediated approaches, and sortase, face challenges including low yields, structural perturbations, and inefficiencies in protein folding, especially with larger proteins, and are not compatible with all expression systems like yeast and mammalian systems.

Innovation Solution

A proximity-based sortase-mediated protein purification and ligation method using a two-construct system where sortase and the protein of interest are on separate fusion proteins, allowing for calcium-triggered release and conjugation of a peptide or protein with an N-terminal glycine, enabling efficient and site-specific conjugation while maintaining protein folding and compatibility with various expression systems.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If sortase is fused directly to the protein of interest in a single-construct system, then conjugation can occur, but protein folding is disrupted and yields are low

Engineering Contradiction:

Improveconjugation capabilityVSAvoidprotein folding

Core Design Contradiction:

Ease of manufactureVSReliability

Solution Approach 1:

The invention divides the single-construct system into two separate constructs: one expressing the protein of interest with a sortase recognition sequence, and another expressing sortase fused to a binding partner. This segmentation allows proper folding of the protein of interest while enabling subsequent conjugation through the sortase-mediated reaction between the recognition sequence and the sortase enzyme.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention introduces a binding partner as an intermediary that mediates the interaction between sortase and the protein of interest. The sortase is fused to the binding partner, creating a complex that can specifically recognize and bind to the protein of interest through the binding partner-protein interaction, thereby enabling conjugation without direct fusion of sortase to the protein.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of manufacture

If traditional conjugation methods are used, then cargo can be attached to protein, but the results are heterogeneous with variable number and location of drugs

Engineering Contradiction:

Improveconjugation capabilityVSAvoidconjugate homogeneity

Core Design Contradiction:

Ease of manufactureVSManufacturing precision

Solution Approach 1:

The invention introduces site-specificity by placing the sortase recognition sequence at a defined location on the protein of interest. This ensures that conjugation occurs only at this specific site rather than randomly throughout the protein, resulting in homogeneous conjugates with uniform drug location and stoichiometry.

Inventive Principle:

Principle #3Local quality

3Manufacturing precision

If sortase-mediated conjugation is used, then site-specific labeling is achieved, but yields are low and the method is not compatible with yeast and mammalian expression systems

Engineering Contradiction:

Improvesite-specific labelingVSAvoidconjugation yield

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The invention enhances compatibility by using a binding partner system that can function across multiple expression systems including yeast and mammalian cells. The binding partner-protein interaction provides a universal mechanism for bringing sortase close to the protein of interest, enabling site-specific conjugation to work effectively in diverse biological systems rather than being limited to bacterial expression only.

Inventive Principle:

Principle #6Universality (Multi-functionality)

4Ease of manufacture

If single-construct sortase system is used, then conjugation occurs, but purification and conjugation are separate steps reducing efficiency

Engineering Contradiction:

Improveconjugation capabilityVSAvoidprocess efficiency

Core Design Contradiction:

Ease of manufactureVSLoss of time

Solution Approach 1:

The invention combines purification and conjugation into a single integrated process. The binding partner is expressed as a fusion with sortase and is immobilized on affinity resin, allowing the protein of interest to be purified through affinity binding while simultaneously positioned for sortase-mediated conjugation. This merging of steps eliminates the need for separate purification and conjugation operations, improving overall process efficiency.

Inventive Principle:

Principle #5Merging (Combining)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This method achieves high ligation efficiency (>95%) and purity of protein conjugates, avoiding structural disruptions and system compatibility issues, and allows for site-specific conjugation of therapeutic and imaging agents to proteins.

Implementation Method 1

sortase-mediated protein purification and ligation

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 2

calcium-triggered release and conjugation

Methodology Applied

Scientific EffectCalcium-triggered release:

Implementation Method 3

the first binding pair partner and the second binding pair partner comprise two protein moieties that form a first heterodimer

Methodology Applied

Scientific EffectProtein-protein interaction:

Data Source

PatentUS12187809B2Proximity-based sortase-mediated protein purification and ligation

Publication Date: 2025.01.07 THE TRUSTEES OF THE UNIV OF PENNSYLVANIA

AI summary

The invention relates to proximity-based sortase-mediated protein purification and ligation. Specifically, the invention relates to techniques that links protein expression/purification with conjugation to therapeutic agents, imaging agents, or linkers.