Phage-Yeast Vector System for Antibody Library Transfer

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Solution Overview

Problem

Current biotechnology methods for protein and antibody display, such as ribosome display, phage display, and yeast display, face limitations including instability of RNA complexes, aggregation issues, and limited transformation efficiency, which restrict the size and diversity of protein libraries that can be effectively screened for antigen binding.

Innovation Solution

Development of vector constructs with specific expression cassettes containing leader sequences, cloning regions with selected restriction sites, and recombinant tags, enabling the transfer and expression of large and diverse protein gene libraries between phage and yeast display systems, enhancing compatibility and complementarity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Quantity of substance

If phage display system is used, then large library size can be accommodated, but proper protein folding and post translational modifications are limited due to prokaryotic expression system

Engineering Contradiction:

Improvelibrary sizeVSAvoidprotein folding quality

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

The invention divides the display system into two separate systems: phage display for maintaining large library size and yeast display for ensuring proper protein folding and post-translational modifications. This segmentation allows each system to optimize for its strength while the combined approach overcomes individual limitations.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention introduces yeast as an intermediary system that receives antibody genes from phage display and performs eukaryotic expression with proper folding and post-translational modifications. The yeast display system acts as a mediator that bridges the gap between prokaryotic library generation and eukaryotic protein quality requirements.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If yeast display system is used, then proper protein folding and post translational modifications are achieved, but transformation efficiency is limited restricting library size

Engineering Contradiction:

Improveprotein folding qualityVSAvoidlibrary size

Core Design Contradiction:

ReliabilityVSQuantity of substance

Solution Approach 1:

The invention merges phage display and yeast display systems into a unified workflow where phage display generates and maintains large libraries, and selected clones are transferred to yeast display for validation of proper folding and function. This combination allows the system to achieve both large library capacity and high protein quality.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

Inventive Principle:

Principle #24Intermediary (Mediator)

3Reliability

If ribosome display method is used, then intracellular expression is achieved, but RNA and ribosomal complex instability limits the system reliability

Engineering Contradiction:

Improveexpression system stabilityVSAvoidtechnical challenge level

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The invention extracts the target protein from the unstable ribosome-RNA complex and displays it on the cell surface using a stable carrier protein (Aga2p). This separates the protein of interest from the unstable molecular machinery, allowing stable display while maintaining the ability to screen large libraries.

Inventive Principle:

Principle #2Taking out (Extraction)

4Reliability

If intracellular selection methods are used, then yeast-two-hybrid or protein complementation can be performed, but aggregation and low cellular half-life reduce screening efficiency

Engineering Contradiction:

Improveselection method capabilityVSAvoidscreening efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The invention extracts the target protein from the intracellular environment and displays it on the cell surface. This removes the protein from the aggregation-prone intracellular milieu and places it in a stable extracellular environment, eliminating aggregation issues and extending cellular half-life while maintaining selection capability.

Inventive Principle:

Principle #2Taking out (Extraction)

Data Source

PatentUS11549119B2Vectors for cloning and expression of proteins, methods and applications thereof

Publication Date: 2023.01.10 ZUMUTOR BIOLOGICS INC

AI summary

The present disclosure relates to vectors for cloning and expressing genetic material including but not limiting to antibody gene or parts thereof and methods of generating said vectors. Said vectors express the antibody genes in different formats such as Fab or scFv as a part of intertransfer system, intratransfer system or direct cloning and expression in individual display systems. In particular, phage display technology is used to clone and screen potential antibody genes in phagemid which is followed by the transfer of said genes to yeast vector for further screening and identification of lead molecules against antigens. The present vectors have numerous advantages including uniquely designed inserts/expression cassettes resulting in efficient and smooth transfer of clonal population from phage to yeast vectors resulting in efficient library preparation and identification of lead molecules.