Preparation of target peptides and proteins
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Solution Overview
Problem
Existing methods for purifying recombinant peptides and proteins are costly, time-consuming, and often result in impurities due to the use of affinity chromatography, chemical cleavage, or chromatography based on size and electrostatic properties, which are not specific and difficult to scale up.
Innovation Solution
A fusion polypeptide comprising a calcium-responsive purification domain and a cleavage domain, allowing for efficient and selective precipitation and intrinsic cleavage to release the target peptide or protein, eliminating the need for chromatographic steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If affinity chromatography is used for purification, then purification efficiency is improved, but production costs increase and time consumption increases
Solution Approach 1:
The invention changes the physical-chemical parameters of the purification process by using calcium-induced precipitation instead of affinity-based binding. The RTX motif undergoes conformational changes upon calcium binding, leading to precipitation that can be easily separated by centrifugation, thereby reducing costs and time while maintaining high purification efficiency
Solution Approach 2:
The invention replaces the complex mechanical affinity chromatography system with a simpler precipitation-based separation system. Instead of using immobilized ligands and column chromatography, the RTX motif is engineered to precipitate in response to calcium, allowing separation through simple centrifugation or filtration
2Productivity
If affinity chromatography is used for purification, then purification efficiency is improved, but time consumption increases
Solution Approach 1:
The invention changes the kinetic parameters of the purification process. Calcium binding to the RTX motif triggers rapid conformational changes and precipitation, which occurs much faster than affinity chromatography steps. This reduces the overall purification time while maintaining high efficiency
Solution Approach 2:
The invention replaces time-consuming affinity chromatography operations with rapid calcium-induced precipitation. The precipitation process can be completed in minutes rather than hours, significantly reducing time consumption while achieving equivalent or superior purification efficiency
3Productivity
If conventional purification domains are used, then purification is achieved, but the purification domain remains with the target peptide after purification
Solution Approach 1:
The invention segments the fusion protein into distinct functional modules: the RTX motif for purification and the target peptide for application. The self-cleaving intein acts as a molecular scissor that automatically separates these segments after purification, ensuring the target peptide is obtained in authentic form without contamination from the purification domain
Solution Approach 2:
The intein domain provides self-service functionality by automatically catalyzing its own excision from the fusion protein after the RTX motif has performed its purification function. This self-cleaving mechanism eliminates the need for additional proteolytic steps and ensures complete removal of the purification domain, delivering authentic target peptide
4Productivity
If external proteases are used for cleavage, then cleavage efficiency is improved, but production costs increase tremendously
Solution Approach 1:
The intein domain provides self-service functionality by automatically catalyzing its own excision from the fusion protein after the RTX motif has performed its purification function. This self-cleaving mechanism eliminates the need for additional proteolytic steps and ensures complete removal of the purification domain, delivering authentic target peptide
5Manufacturing precision
If autoproteases are used for cleavage, then authentic peptides are obtained, but unintended activation during purification leads to loss in yield
Solution Approach 1:
The invention segments the fusion protein into distinct functional modules: the RTX motif for purification and the target peptide for application. The self-cleaving intein acts as a molecular scissor that automatically separates these segments after purification, ensuring the target peptide is obtained in authentic form without contamination from the purification domain
Solution Approach 2:
The purification step using calcium-induced precipitation is performed as a preliminary action before cleavage. This sequence ensures that the fusion protein is fully purified and concentrated before the intein is activated for cleavage, preventing premature cleavage and maximizing yield while still achieving authentic peptide products
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
Enables highly efficient purification with authentic termini at low costs and high yields, independent of protein nature, using calcium-induced precipitation and autoproteolysis or chemical cleavage.
Implementation Method 1
a purification domain, wherein the purification domain comprises 3 to 50 repeats of a nonapeptide... calcium-responsive, reversible precipitation
Implementation Method 2
a cleavage domain, wherein the cleavage domain comprises an autoprotease... intrinsic cleavage by autoproteolysis
Data Source
AI summary
The present invention relates to a fusion polypeptide comprising a purification domain; a cleavage domain; and a target peptide domain. The purification domain comprises 3 to 50 repeats of a nonapeptide, each nonapeptide independently having an amino acid sequence GX2X3X4X5X6X7X8X9, whereby X2 may be G, N or D, X3 may be A, S, G, D, E, L or N, X4 may be G or A, X5 may be N, D, A or S, X6 may be D or N, X7 may be T, I, V or L, X8 may be L, V, I, F or Y, and X9 may be Y, I, V, F, T, N, D, K or S. The cleavage domain comprises an autoprotease or an amino acid sequence XaSXbXcXdXeXfXg, whereby Xa is only optional and may be any amino acid, Xb may be H, R, S, N, Y, G, K or Q, Xc may be H, S or N, Xd may be T, S, L, M, G, N, Q or W, Xe is only optional may be S, P, A, T, L, R or D, Xf is only optional and may be L, P, E, G, T, A, F or S, and Xg is only optional and may be any amino acid. Further disclosed is a method for preparing a target peptide using the fusion polypeptide of the invention.