Collagen Protein Secretion in C. Glutamicum Without Protease Cleavage
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Solution Overview
Problem
Existing methods for producing collagen proteins and bacterial collagen-like proteins are costly due to the need for specific proteases to cleave the V domain, and the process is hindered by the separation of the protein from intracellular fractions and the purification of immature CLP, which complicates the workflow.
Innovation Solution
Utilizing Corynebacterium glutamicum as a host for secretion, employing specific signal peptides that enhance the secretion efficiency and selectivity of collagen proteins and bacterial collagen-like proteins, allowing for high-level production without the need for additional protease cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If E. coli is used to express collagen-like proteins with protease cleavage site modification, then the V domain can be removed, but the manufacturing cost increases due to expensive proteases and complex purification
Solution Approach 1:
The invention extracts and removes the problematic V domain through genetic modification at the DNA level rather than requiring post-expression protease treatment. The modified gene sequence directly produces mature collagen-like protein without the V domain, eliminating the need for expensive protease enzymes and simplifying purification processes.
Solution Approach 2:
The invention performs the domain removal action in advance during gene construction rather than after protein expression. By designing the expression construct with the V domain already excluded from the coding sequence, the mature protein is produced directly in its final form, preventing the need for subsequent protease treatment and purification steps.
2Productivity
If intracellular expression is used, then protein production occurs, but separation from intracellular fractions becomes difficult and costly
Solution Approach 1:
The invention extracts the collagen-like protein from the intracellular compartment by implementing a secretion signal sequence at the N-terminus of the expression construct. This directs the protein to be secreted into the extracellular medium, automatically separating it from intracellular fractions and simplifying purification while maintaining high production levels.
3Manufacturing precision
If protease cleavage is used to remove V domain, then immature CLP is processed, but additional purification steps are required
Solution Approach 1:
The invention performs the domain removal action in advance during gene construction rather than after protein expression. By designing the expression construct with the V domain already excluded from the coding sequence, the mature protein is produced directly in its final form, preventing the need for subsequent protease treatment and purification steps.
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
Achieves high-level secretion and purity of collagen proteins, reducing manufacturing costs by eliminating the need for protease cleavage and improving the authenticity of the amino acid sequence, suitable for medical and pharmaceutical applications.
Implementation Method 1
Corynebacterium glutamicum (C. glutamicum) has been used in the past in many instances to produce heterologous proteins via secretion into the supernatant
Data Source
AI summary
Polynucleotides with an N-terminal signal sequence encode an amino acid sequence encoding a collagen protein or a bacterial collagen-like protein. Fusion products of collagen-like protein with various N-terminal signal peptides lead to increased production of collagen-like protein and secretion in a fermentative process for secreting bacterial collagen-like proteins in a host.