Gram-positive bacterial pili tip polypeptide display
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Solution Overview
Problem
Current methods for displaying heterologous proteins on the surface of microorganisms, such as yeast and bacteria, are limited in their ability to effectively expose proteins of interest for immunization, bioremediation, and other applications, as they often fail to ensure proper folding and maximize exposure to the immune system.
Innovation Solution
The method involves introducing a polynucleotide encoding a chimeric polypeptide into Gram-positive bacteria, which includes a protein of interest linked to a Gram-positive bacterial pilus tip protein with a cell wall sorting signal, allowing the protein to be covalently attached to the tip of pili, thereby enhancing exposure and proper folding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If heterologous proteins are displayed on the surface of microorganisms using conventional methods, then the proteins can be exposed for immunization and bioremediation, but the proteins often fail to achieve proper folding and maximum exposure to the immune system
Solution Approach 1:
The patent uses a pilus tip protein as an intermediary carrier to display the heterologous protein of interest. The pilus tip protein serves as a mediator that facilitates proper folding and optimal exposure of the heterologous protein on the bacterial surface, resolving the contradiction between reliable protein folding and ease of manufacture/exposure.
Solution Approach 2:
The patent employs a cell wall sorting signal (CWSS) that directs the chimeric polypeptide to the cell wall before final protein folding and display occur. This preliminary sorting action ensures proper localization and folding conditions are established early in the process, enabling subsequent optimal exposure of the heterologous protein.
2Ease of operation
If heterologous proteins are displayed on the surface of microorganisms, then immunization and bioremediation applications can be pursued, but the current methods fail to maximize exposure to the immune system
Solution Approach 1:
The patent displays the heterologous protein specifically at the tip of pili, which are surface structures that extend from the bacterial cell. This localized display at the pilus tip maximizes exposure to the immune system while maintaining proper protein folding and function, as the pilus tip provides a favorable local environment for protein expression.
Solution Approach 2:
The patent extends the display of heterologous proteins from the general bacterial surface into the third dimension by using pilus structures that protrude from the cell surface. This dimensional extension maximizes exposure to the immune system while maintaining proper protein folding through the structured environment of the pilus.
3Manufacturing precision
If a chimeric polypeptide is constructed with a cell wall sorting signal to direct protein to the cell wall, then proper localization and folding are achieved, but the system requires multiple components including tip sortase and pilus shaft polypeptide
Solution Approach 1:
The cell wall sorting signal (CWSS) is a universal signaling sequence that directs multiple different chimeric polypeptides to the cell wall through the same sortase-mediated pathway. This universal sorting mechanism simplifies the system by using a common localization pathway for different protein displays, reducing the need for protein-specific localization components.
Solution Approach 2:
The patent divides the pilus structure into functional segments: the pilus shaft polypeptide forms the structural backbone, the tip sortase provides the enzymatic function for attachment, and the CWSS provides the targeting signal. This segmentation allows each component to perform its specialized function efficiently while maintaining overall system coordination for precise protein localization.
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 effectively displays the protein of interest on the bacterial surface, maximizing immunological response and bioremediation capabilities by ensuring proper folding and increased exposure, enhancing the odds of functional protein expression.
Implementation Method 1
The chimeric polypeptide comprises the polypeptide of interest and a Gram-positive bacterial pilus tip protein... The Gram-positive bacterium expressing the chimeric polypeptide also expresses a tip sortase... allowing the protein to be covalently attached to the tip of pili
Data Source
AI summary
Provided herein are methods and compositions for the display of polypeptides of interest on the tip of pili of Gram-positive bacteria. According to the present invention, the polypeptide of interest is amino terminal to a Gram-positive bacterial pilus tip protein or an active variant or fragment thereof, wherein the active variant or fragment comprises a cleaved cell wall sorting signal (CWSS) motif. The Gram-positive bacterium displaying a polypeptide of interest on the tip of pili that are disclosed herein are useful, for example, in methods for immunizing a subject with an antigen and methods for removing contaminants from a composition.