Periplasmic Phage-Assisted Continuous Evolution for Disulfide Proteins
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current continuous evolution methods are limited in evolving proteins that require a non-reducing environment for proper folding and function, particularly those with disulfide bonds, as the cytoplasmic expression in bacteria does not support disulfide linkage formation, leading to protein instability and aggregation.
Innovation Solution
The method involves expressing proteins of interest in the bacterial periplasm using phage-assisted continuous evolution (PACE) by encoding the protein in a bacteriophage genome and using a periplasmic capture agent to activate a conditional promoter for pIII expression, allowing disulfide bond formation and linking protein activity to phage propagation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If proteins are expressed in the bacterial cytoplasm using conventional PACE methods, then the evolution process can proceed continuously with minimal intervention, but disulfide bond formation is inhibited leading to protein instability and aggregation
Solution Approach 1:
The invention divides the bacterial cell into two functional compartments: the cytoplasm for continuous phage replication and evolution, and the periplasm for proper protein folding and disulfide bond formation. This spatial segmentation allows proteins to be expressed in the oxidizing periplasmic environment where disulfide bonds can form correctly, while maintaining the continuous evolution capability of cytoplasmic PACE systems.
Solution Approach 2:
The invention introduces a periplasmic capture agent as an intermediary component that activates a conditional promoter to drive pIII expression. This mediator links the protein's desired activity in the periplasm to phage propagation, enabling selection of functional proteins that require disulfide bonds while maintaining continuous evolution.
2Reliability
If thiol oxidase and disulfide isomerase are expressed in the cytoplasm to support disulfide bond formation, then disulfide-containing proteins can fold properly, but cellular stress increases leading to membrane impairment and aggregation
Solution Approach 1:
The invention extracts the oxidative environment required for disulfide bond formation from the cytoplasm and relocates it to the periplasm, where oxidizing conditions naturally exist. This eliminates the need to introduce non-native oxidative enzymes into the cytoplasm, thereby avoiding the cellular stress and membrane impairment that would result from such interventions.
Solution Approach 2:
The invention changes the environmental parameters (oxidizing conditions) by expressing the protein of interest in the periplasm rather than the cytoplasm. This parameter change allows disulfide bonds to form naturally without introducing foreign enzymes, avoiding the harmful effects of oxidative stress on the bacterial cell.
3Reliability
If compensatory stabilizing mutations are introduced to maintain protein activity in the reducing cytoplasm, then proteins can remain biologically active, but target affinity and other biological functions are compromised
Solution Approach 1:
The invention inverts the conventional approach by not trying to adapt proteins to the reducing cytoplasmic environment, but rather expressing them in the oxidizing periplasmic environment where they can fold correctly with their native disulfide bonds. This eliminates the need for compensatory mutations that would otherwise be required to maintain activity in the cytoplasm, preserving both protein activity and target affinity.
4Productivity
If binding affinity evolution is performed in the reducing cytoplasm, then evolution can proceed continuously, but disulfide-containing extracellular antigens cannot be effectively targeted
Solution Approach 1:
The invention segments the evolution system to express proteins in the periplasm where they can properly fold with disulfide bonds, enabling them to bind disulfide-containing extracellular antigens. Simultaneously, the cytoplasmic compartment maintains continuous phage replication and evolution throughput, achieving both goals through spatial division.
Solution Approach 2:
The periplasmic capture agent serves as an intermediary that enables the selection of proteins capable of binding extracellular antigens. By positioning the capture agent and target antigen interaction in the periplasmic environment, the system can evolve proteins with authentic disulfide bonds that maintain their ability to bind extracellular targets while preserving continuous evolution throughput.
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 enables the evolution of proteins that require oxidizing conditions for stability and function, improving protein stability and solubility while maintaining desired biological activities, facilitating the development of therapeutic proteins.
Implementation Method 1
The bacterial periplasm, which is an oxidizing environment, supports the formation of disulfides in proteins
Implementation Method 2
An accessory plasmid (AP) within a host E. coli cell expresses gIII under the control of a transcriptional circuit that is activated in response to the desired function of the evolving protein
Implementation Method 3
As phage depend on pIII, the protein product of gIII, to efficiently infect host cells, PACE links the desired property of an evolving protein with the ability of the phage that encodes it to replicate
Data Source
AI summary
Aspects of the disclosure relate to compositions, systems, and methods for evolving nucleic acids and proteins utilizing continuous directed evolution in the periplasm of a host cell. In some embodiments, the methods comprise passing a nucleic acid from cell-to-cell in a desired, function dependent manner. The linkage of the desired function and passage of the nucleic acid from cell-to-cell allows for continuous selection and mutation of the nucleic acid.