Compatible Polynucleotide Vector Systems for Library Transfer

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Solution Overview

Problem

Current methods for high-throughput screening of polynucleotides encoding protein variants, such as phage display techniques, are limited by bacterial transformation efficiency and compatibility issues between different vector systems, restricting the seamless conversion and optimization of antigen binding proteins for properties like affinity, stability, and specificity.

Innovation Solution

A polynucleotide vector system with compatible restriction sites allows for the transfer of polynucleotides between ribosome display and phage display systems using a restriction enzyme digestion and ligation strategy, enabling the generation and conversion of large protein libraries for affinity maturation and expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If phage display techniques are used for high-throughput screening, then large libraries of polynucleotides can be screened, but bacterial transformation efficiency limits the library size and compatibility between vector systems

Engineering Contradiction:

Improvelibrary sizeVSAvoidbacterial transformation efficiency

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The patent introduces ribosome display as an intermediary system between phage display and final protein expression. Ribosome display uses in vitro translation to display proteins on ribosomes, eliminating dependence on bacterial transformation efficiency while maintaining the ability to screen large libraries. The ribosome display system serves as a bridge to transfer successful clones to phage display or expression systems.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent replaces the mechanical process of bacterial transformation with in vitro translation for protein display. Instead of relying on bacteria to transform and express proteins (phage display), the system uses purified ribosomes and translation factors to synthesize and display proteins in a test tube (ribosome display), thereby eliminating the bottleneck of bacterial transformation efficiency.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Adaptability or versatility

If different vector systems are used for ribosome display and phage display, then each system can be optimized for its specific function, but compatibility issues prevent seamless conversion between systems

Engineering Contradiction:

Improvesystem optimizationVSAvoidconversion between vector systems

Core Design Contradiction:

Adaptability or versatilityVSEase of manufacture

Solution Approach 1:

The patent segments the polynucleotide into distinct functional regions with standardized restriction sites. The coding sequence is flanked by specific restriction sites (e.g., NcoI at the 5' end and XhoI at the 3' end) that are compatible across different vector systems. This segmentation allows the coding region to be independently transferred between ribosome display, phage display, and expression vectors without redesigning the entire construct.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent creates a universal polynucleotide backbone that can function in multiple different vector systems. By incorporating standardized restriction sites and compatible cloning elements, the same basic polynucleotide structure can be used in ribosome display vectors, phage display vectors, and expression vectors, enabling seamless conversion and eliminating the need for system-specific redesign.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Productivity

If large protein libraries are generated for affinity maturation, then more variants can be screened for improved binding properties, but the complexity of managing and converting between different vector systems increases

Engineering Contradiction:

Improvescreening throughputVSAvoidvector system conversion

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent performs preliminary action by pre-installing standardized restriction sites and compatible cloning elements during the initial polynucleotide construction. This preliminary preparation ensures that subsequent transfers between different vector systems can be performed using simple, standardized procedures rather than requiring complex system-specific protocols for each library conversion.

Inventive Principle:

Principle #10Preliminary action

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 approach facilitates the efficient optimization of antigen binding proteins by allowing the transfer of large protein libraries between vector systems, overcoming previous limitations and enabling the identification of variants with improved binding affinity and specificity.

Implementation Method 1

a polynucleotide vector system with compatible restriction sites allows for the transfer of polynucleotides between ribosome display and phage display systems using a restriction enzyme digestion and ligation strategy

Methodology Applied

Scientific EffectRestriction enzyme digestion: Enzyme

Implementation Method 2

Methodology Applied

Scientific EffectLigation: Chemical Bonding

Data Source

PatentUS9523092B2Compatible display vector systems

Publication Date: 2016.12.20 WYETH LLC

AI summary

The present invention provides a polynucleotide vector system used during polypeptide display that can be used to facilitate transfer of pools of polynucleotides encoding antigen binding proteins of interest. The present invention also provides methods that allow seamless conversion of pools of polynucleotides encoding antigen binding proteins using a restriction enzyme digestion and ligation strategy.