Modular Polypeptide Expression System via Trans-Splicing

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Solution Overview

Problem

Current polypeptide expression systems require multiple constructs for each combination of recurring modules, leading to a geometric increase in the number of constructs needed, which is resource-intensive and generates unnecessary constructs, especially when expressing large protein collections.

Innovation Solution

A polypeptide expression system comprising two nucleic acid molecules with specific components, including eukaryotic and prokaryotic promoters, splice sites, and hybridizing sequences, allowing for modular expression and production through trans-splicing in eukaryotic cells, enabling the precise joining of protein-coding sequences without the need for subcloning.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If traditional recombinant DNA methods are used to join sequences encoding each module, then each polypeptide combination requires a separate construct, but the number of constructs increases geometrically leading to resource intensiveness and generation of unnecessary constructs

Engineering Contradiction:

Improveease of constructing polypeptide expression systemsVSAvoidnumber of constructs required

Core Design Contradiction:

Ease of manufactureVSDevice complexity

Solution Approach 1:

The invention divides the polypeptide expression system into modular components: a core nucleic acid molecule containing essential elements (promoter, splice sites, hybridizing sequence) and separate module-specific nucleic acid molecules encoding different polypeptide domains. This segmentation allows independent assembly of modules through trans-splicing rather than requiring separate complete constructs for each combination.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The core nucleic acid molecule serves as a universal platform that can combine with multiple different module-specific nucleic acid molecules through complementary hybridizing sequences. This multi-functional core structure enables a single construct to support expression of numerous polypeptide combinations, eliminating the need for geometrically increasing numbers of specialized constructs.

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Productivity

If high-throughput systems for subcloning are used to handle large number of inserts in parallel, then processing capacity increases, but resource consumption increases and large number of unnecessary constructs are generated

Engineering Contradiction:

Improveprocessing capacity for polypeptide expressionVSAvoidresource consumption

Core Design Contradiction:

ProductivityVSLoss of energy

Solution Approach 1:

The core nucleic acid molecule is pre-designed with universal elements including promoter regions, splice sites, and hybridizing sequences before combination with module-specific sequences. This preliminary preparation of the core structure eliminates the need for time-consuming subcloning operations and reduces resource consumption during the actual assembly process.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The hybridizing sequence acts as an intermediary element that facilitates specific pairing between the core nucleic acid molecule and module-specific nucleic acid molecules. This mediator mechanism enables precise, automated assembly without requiring labor-intensive subcloning procedures, thereby increasing productivity while reducing resource usage.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Reliability

If multiple constructs are created for initial characterization steps, then comprehensive characterization is achieved, but many constructs are ultimately not necessary after characterization

Engineering Contradiction:

Improvecharacterization accuracy of polypeptide expressionVSAvoidnumber of constructs produced

Core Design Contradiction:

ReliabilityVSQuantity of substance

Solution Approach 1:

The system allows dynamic selection and combination of module-specific nucleic acid molecules with the core structure based on characterization requirements. Rather than statically creating all possible constructs, the modular design enables flexible assembly of only those combinations needed for specific characterization purposes, reducing the total number of constructs while maintaining characterization reliability.

Inventive Principle:

Principle #15Dynamics

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 system simplifies the expression of large protein collections by reducing the number of constructs required, increasing efficiency and reducing resource consumption, while allowing for flexible expression of different antibody formats and formats without the need for labor-intensive subcloning.

Implementation Method 1

the first nucleic acid molecule further comprises a hybridizing sequence (HS1), and the second nucleic acid molecule comprises a hybridizing sequence capable of hybridizing to HS1 (HS2)

Methodology Applied

Scientific EffectHybridization:

Data Source

PatentUS11851661B2Polypeptide expression systems

Publication Date: 2023.12.26 GENENTECH INC

AI summary

The present invention relates to polypeptide expression systems and methods of using the same.