Circularly Permuted Carrier Polypeptides for Protease-Resistant Bacterial Display

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Solution Overview

Problem

Existing bacterial polypeptide display systems face challenges such as limited expression of large peptides, inability to display diverse sequences, adverse effects on cell growth, and protease sensitivity, particularly in complex environments.

Innovation Solution

The use of carrier polypeptides (CPs) like CYTX-CPs, which are circularly permuted beta barrel-shaped transmembrane polypeptides with protease-resistant sequences and flexible linkers, to display polypeptides on the outer surface of bacteria, enhancing resistance to protease degradation and improving display characteristics.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Length of moving object

If conventional bacterial display systems are used, then small peptides can be expressed, but large polypeptides cannot be properly displayed

Engineering Contradiction:

Improvepolypeptide lengthVSAvoiddisplay stability

Core Design Contradiction:

Length of moving objectVSReliability

Solution Approach 1:

The system segments the polypeptide display function across multiple components: the circularly permuted transmembrane protein provides structural stability and membrane anchoring, while the extracellular N- and C-termini serve as dedicated display regions. This segmentation allows large polypeptides to be displayed without compromising the stability of the core transmembrane structure.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention changes key parameters of the display system by using circularly permuted transmembrane proteins with specific extracellular loop structures and flexible linker regions. These parameter changes enable the system to accommodate polypeptides of varying lengths while maintaining proper display orientation and stability.

Inventive Principle:

Principle #35Parameter changes

2Ease of operation

If polypeptides are displayed on the outer membrane surface, then accessibility is improved, but protease sensitivity increases

Engineering Contradiction:

Improvesurface accessibilityVSAvoidprotease degradation

Core Design Contradiction:

Ease of operationVSObject-affected harmful factors

Solution Approach 1:

The system employs flexible linker regions and optimized extracellular loop structures that act as protective cushions between the displayed polypeptide and proteolytic enzymes. These structural elements reduce protease accessibility while maintaining the displayed polypeptide's exposure for ligand binding.

Inventive Principle:

Principle #11Beforehand cushioning (Prior cushioning)

Solution Approach 2:

The extracellular loops and flexible linkers of the circularly permuted transmembrane protein form a protective structural shell around the displayed polypeptide. This flexible shell provides physical protection against protease degradation while allowing the displayed polypeptide to remain accessible for its functional interactions.

Inventive Principle:

Principle #30Flexible shells and thin films

3Adaptability or versatility

If diverse polypeptide sequences are displayed, then library diversity is enhanced, but cell growth is adversely affected

Engineering Contradiction:

Improvesequence diversityVSAvoidcell growth rate

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The circularly permuted transmembrane protein serves multiple functions: it provides structural stability in the membrane, enables diverse polypeptide display through its extracellular termini, and maintains cell viability through its stable folding and proper membrane integration. This multi-functionality allows diverse sequence libraries to be displayed without compromising cell growth.

Inventive Principle:

Principle #6Universality (Multi-functionality)

4Ease of manufacture

If traditional expression vectors are used, then simple construction is achieved, but limited polypeptide sizes can be expressed

Engineering Contradiction:

Improvevector construction simplicityVSAvoidexpressed polypeptide size

Core Design Contradiction:

Ease of manufactureVSLength of moving object

Solution Approach 1:

The invention transitions from traditional periplasmic or cytoplasmic expression to outer membrane surface display through circularly permuted transmembrane proteins. This dimensional change from intracellular to extracellular display enables the expression of larger polypeptides that would be problematic in traditional systems, while maintaining relatively simple vector construction based on established bacterial display platforms.

Inventive Principle:

Principle #17Another dimension (Dimensionality change)

Data Source

PatentUS20250034755A1Protease-resistant systems for polypeptide display and methods of making and using thereof

Publication Date: 2025.01.30 CYTOMX THERAPEUTICS INC

AI summary

The present invention generally relates to bacterial polypeptide display systems, libraries using these bacterial display systems, and methods of making and using these systems, including methods for improved display of polypeptides on the extracellular surface of bacteria using circularly permuted transmembrane bacterial polypeptides that have been modified to increase resistance to protease degradation and to enhance polypeptide display characteristics.