Recombinant Collagen-like Protein Purification via Solvent Precipitation
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Solution Overview
Problem
Current methods for producing and purifying recombinant collagen-like proteins, such as the triple helical Scl2 protein, are costly and inefficient due to the need for complex downstream processes, including protease usage and cell lysis, which are hindered by the immunogenic V-domain, and achieve low purity due to the solubility challenges in water-miscible organic solvents.
Innovation Solution
A novel method involving fermentation of host cells expressing a collagen-like protein, followed by incubation for folding and subsequent purification using solvent precipitation with organic solvents like 2-propanol, ethanol, or acetone, which allows for high-purity extraction without the need for protease digestion and cell lysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If the V-domain is retained in the collagen-like protein construct, then the protein can be folded into a triple helical structure, but the protein cannot be transported out of the host cell and requires complex cell lysis for extraction
Solution Approach 1:
The collagen-like protein is divided into two functional segments: the V-domain and the collagen-like domain (CLD). By removing the V-domain, the patent segments the protein to eliminate the portion causing transport issues while retaining the functional CLD portion that forms the triple helical structure.
Solution Approach 2:
The V-domain is extracted and removed from the collagen-like protein construct. This extraction eliminates the harmful effect of the V-domain (preventing cell export) while preserving the beneficial CLD portion that self-folds into the desired triple helical structure.
2Object-affected harmful factors
If protease is used to remove the V-domain, then the immunogenic V-domain is eliminated, but the process becomes costly and requires additional purification steps to remove the protease
Solution Approach 1:
The V-domain is extracted at the genetic level by designing a construct that only contains the collagen-like domain without the V-domain sequence. This eliminates the need for protease digestion entirely, as the V-domain is never present in the expressed protein.
Solution Approach 2:
The V-domain removal is performed preliminarily during construct design rather than during downstream processing. By creating a V-domain-free construct before expression, the patent avoids the need for protease treatment and subsequent protease removal steps.
3Quantity of substance
If conventional purification methods are used, then the protein can be extracted from the fermentation broth, but the purity remains low due to solubility challenges in water-miscible organic solvents
Solution Approach 1:
The patent changes the solvent parameter from water-miscible organic solvents (which maintain high protein solubility) to water-immiscible organic solvents (which decrease protein solubility). This parameter change causes the collagen-like protein to precipitate selectively, enabling high-purity recovery.
Solution Approach 2:
The patent utilizes phase transition by adding water-immiscible organic solvents to induce protein precipitation. The protein transitions from a soluble state in the aqueous fermentation broth to an insoluble precipitated state, allowing for easy separation and high-purity recovery through centrifugation or filtration.
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 method achieves high purity (>75%) of collagen-like proteins by leveraging the decreased solubility of the protein in organic solvents, reducing costs and simplifying the purification process, and eliminating the need for expensive proteases and complex cell handling.
Implementation Method 1
purification of the CLP by solvent precipitation... purification of triple helical Scl2 protein by precipitation using organic solvents... decreased solubility of the protein in organic solvents
Data Source
AI summary
A method is developed for producing a recombinant collagen-like protein (CLP). The method includes fermenting of a host cell, expressing a polynucleotide encoding an amino acid sequence encoding a CLP, incubating the fermentation broth for at least 1 hour at not more than 25° C. for folding of the CLP, and purifying of the CLP by solvent precipitation.