Pro-polypeptide Construct for Prokaryotic Expression and Endotoxin Removal
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Solution Overview
Problem
Current expression systems for recombinant polypeptides in prokaryotic cells face challenges in achieving high yields and efficient downstream processing, particularly in removing endotoxins and purifying proteins of interest.
Innovation Solution
A pro-polypeptide construct is used, comprising a dipeptide GS sequence, an amino acid tag, and a protease cleavage site, which is fused to the N-terminus of the polypeptide of interest, allowing for improved expression yields and efficient protease-mediated cleavage for purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If polypeptides are produced in prokaryotic cells using conventional expression systems, then production capability is achieved, but expression yields are low and downstream processing is inefficient
Solution Approach 1:
The patent introduces a pro-polypeptide as an intermediary component that facilitates both high-level expression in prokaryotic cells and efficient downstream processing. The pro-polypeptide contains a protease cleavage site that enables specific cleavage to remove endotoxins and纯化 the target polypeptide, thereby resolving the contradiction between achieving high expression yields and maintaining ease of manufacture through efficient downstream processing.
2Ease of operation
If conventional expression systems are used in prokaryotic cells, then simple system operation is maintained, but endotoxin removal is inefficient and purification is difficult
Solution Approach 1:
The patent applies preliminary action by incorporating a protease cleavage site into the pro-polypeptide structure before expression. This pre-designed feature enables subsequent specific protease treatment to efficiently remove endotoxins and simplify purification, thereby resolving the contradiction between maintaining simple system operation and eliminating endotoxin contamination without adding complex operational steps.
3Productivity
If fusion polypeptides are produced with pro-polypeptide, then expression yield is improved, but additional cleavage step is required
Solution Approach 1:
The patent utilizes parameter changes by designing the pro-polypeptide with a specific protease cleavage site that changes the molecular structure at a defined location. This allows specific protease recognition and cleavage under controlled conditions, enabling efficient separation of the target polypeptide from the pro-polypeptide fusion. The cleavage step, while additional, is highly specific and efficient, thereby resolving the contradiction between improved expression yield and processing complexity by making the additional step highly targeted and manageable.
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 enhances expression yields and facilitates efficient endotoxin removal and purification of the polypeptide of interest, improving the overall handling and recovery process.
Implementation Method 1
the pro-polypeptide can efficiently be cleaved from the polypeptide of interest by the incorporated protease cleavage site with the cognate protease
Data Source
AI summary
A pro-polypeptide which is useful for the expression of a polypeptide of interest in a prokaryotic cell. Therefore the pro-polypeptide is fused to the N-terminus of the polypeptide of interest. The pro-polypeptide as reported herein provides for improved expression yields and improves the handling of the fusion polypeptide (downstream processing, purification). For example, efficient endotoxin removal is effected while the protein of interest comprising the pro-polypeptide is bound e.g. to an affinity chromatography material. Thereafter the pro-polypeptide can efficiently be cleaved from the polypeptide of interest by the incorporated protease cleavage site with the cognate protease.