Intein Polypeptide Self-Excision for Native N-Terminal Cleavage
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Solution Overview
Problem
Biotechnological manufacturing faces challenges in removing N-terminal amino acid residues from polypeptides and producing proteins that are toxic to host cells, particularly in achieving specific cleavage of polypeptides and proteins at desired residues.
Innovation Solution
The use of intein polypeptides with specific amino acid sequences for self-excision and cleavage, allowing for the production of proteins with native N-terminal residues and enabling the expression of toxic proteins by incorporating intein sequences that can excise without ligation or with reduced toxicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If intein polypeptides are used for self-excision and cleavage, then proteins with native N-terminal residues are generated, but the complexity of the manufacturing process increases
Solution Approach 1:
The intein polypeptide performs self-excision from the target polypeptide through its intrinsic protein splicing activity, eliminating the need for external enzymes or complex cleavage machinery. The intein autonomously catalyzes its own removal and joins the flanking extein sequences, achieving precise cleavage while simplifying the overall process architecture despite the added molecular complexity of the intein sequence itself
Solution Approach 2:
The intein polypeptide acts as an intermediary element inserted between the N-terminal methionine and the rest of the target protein sequence. This intermediate sequence facilitates controlled cleavage at the desired location while preserving the native N-terminal residue of the target protein, effectively mediating the transition from a complex manufacturing challenge to a manageable biochemical reaction
2Object-affected harmful factors
If intein sequences are incorporated to enable expression of toxic proteins, then host cell toxicity is reduced, but the device complexity increases
Solution Approach 1:
The toxic polypeptide is expressed in advance as a fusion protein with the intein sequence, allowing the host cell to tolerate the initial expression event. The intein-mediated self-excision then occurs subsequently to release the active toxic protein, separating the expression phase (where toxicity would be problematic) from the release phase (where the toxic protein becomes functional but in controlled amounts)
Solution Approach 2:
The intein sequence serves as a protective intermediary that masks the toxicity of the target protein during expression. By incorporating the toxic sequence into an intein-fusion construct, the host cell encounters a benign fusion protein rather than the active toxic protein, thereby reducing cellular damage while still enabling production of the desired toxic product
3Manufacturing precision
If N-terminal methionine residue removal is achieved through intein self-excision, then native N-terminal residues are obtained, but the manufacturing process becomes more complex
Solution Approach 1:
The intein polypeptide autonomously performs the N-terminal methionine removal function through its self-excision capability, eliminating the need for separate enzymatic treatment steps or complex chemical deprotection procedures. The single intein-mediated reaction simultaneously achieves both the removal of unwanted residues and the generation of the native N-terminus, simplifying manufacturing despite the increased molecular complexity of the intein tag
Solution Approach 2:
The intein sequence introduces specific amino acid parameters and sequence features that enable controlled self-excision at the desired location. By carefully designing the intein-extein junction sequences and utilizing the inherent biochemical properties of the intein domain, the method achieves precise N-terminal residue control through parameter optimization rather than complex process 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
This method effectively generates proteins with native N-terminal residues and facilitates the expression of toxic proteins by ensuring specific cleavage and reduced host cell toxicity, enhancing biomanufacturing efficiency.
Implementation Method 1
intein polypeptides...for the cleavage of polypeptides and proteins at specific residues...self-excision and cleavage
Data Source
AI summary
Methods and constructs for the cleavage of polypeptides at one or more specific positions within the polypeptide are provided.