Multivalent Phage Display via pIX Segmentation

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Solution Overview

Problem

Current methods for phage display using pIX protein are limited to mono- or oligovalent display, which is insufficient for applications requiring multivalent display, such as isolating rare and weakly binding clones or stability engineering strategies.

Innovation Solution

The development of vectors and systems that utilize M13 phage pIX protein and helper phage vectors encoding wild-type pIX with suppressor codons, allowing for multivalent display of protein-of-interest:pIX fusion proteins through phagemid rescue in non-suppressor host strains, where the helper phage produces full-length pIX in suppressor hosts and truncated pIX in non-suppressor hosts.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If helper phage encodes wild-type pIX protein, then phage assembly is maintained and virion stability is ensured, but multivalent display of protein-of-interest is prevented

Engineering Contradiction:

Improvephage assembly and virion stabilityVSAvoidmultivalent display capability

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The system segments the pIX protein function by creating two distinct versions: wild-type pIX in helper phage for assembly/stability, and modified pIX (with suppressor codons) in phagemid for multivalent display. This segmentation allows each component to fulfill its specific role without interference.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Suppressor codons act as intermediaries that control protein expression based on host strain. In non-suppressor hosts, the suppressor codons prevent translation of wild-type pIX from helper phage, enabling multivalent display. In suppressor hosts, translation proceeds normally, maintaining assembly and stability.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If pIX is fused with protein-of-interest for display, then multivalent display is achieved, but phage assembly is abolished

Engineering Contradiction:

Improvemultivalent display capabilityVSAvoidphage assembly

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The suppressor codon serves as a conditional intermediary that differentiates between helper phage-derived pIX (with suppressor codons, expressed in non-suppressor hosts) and phagemid-derived pIX (without suppressor codons, not expressed when suppressor codons block translation). This enables selective expression based on host strain.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The system applies different qualities to different sources of pIX: helper phage provides pIX with suppressor codons for conditional expression, while phagemid provides pIX without suppressor codons for constitutive expression in appropriate hosts. This local differentiation resolves the assembly-display conflict.

Inventive Principle:

Principle #3Local quality

3Adaptability or versatility

If suppressor codons are included in helper phage pIX, then multivalent display is enabled in non-suppressor hosts, but production of full-length pIX is prevented

Engineering Contradiction:

Improvemultivalent display capabilityVSAvoidfull-length pIX production

Core Design Contradiction:

Adaptability or versatilityVSQuantity of substance

Solution Approach 1:

The suppressor codons, which would normally be harmful by preventing full-length protein production, are converted into a beneficial tool for controlling expression. In non-suppressor hosts, they prevent production of wild-type pIX that would interfere with multivalent display, while still allowing production of the modified pIX from phagemid.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Data Source

PatentUS10640761B2Multivalent phage display systems and methods

Publication Date: 2020.05.05 UNIVERSITY OF OSLO

AI summary

The present invention relates to vectors, methods and systems for polypeptide display and selection. Specifically, the present invention relates to vectors, methods, and systems for multivalent phage display using pIX protein of filamentous phage and helper phage.