Recombinant Polypeptide Cleavage for Exact Peptide Sequences
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Solution Overview
Problem
Existing recombinant peptide production methods face challenges in producing peptides with exact intended sequences, high yield, and cost-effectiveness, while avoiding extra amino acids that can cause immunogenic responses and interfere with physiological functions, and current methods are not suitable for a wide range of peptide lengths.
Innovation Solution
Employing enzyme-catalyzed and chemical proteolysis to cleave recombinant polypeptides with linked peptides, using proteases that recognize specific cleavage sites and leave no extraneous amino acids, combined with nucleic acid constructs and cell-based systems for efficient production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional recombinant peptide production methods are used, then peptide production is achieved, but extra amino acids are left in the final peptide product which can cause immunogenic responses and interfere with physiological functions
Solution Approach 1:
The patent applies the extraction principle by removing the problematic linker peptide sequence from the recombinant polypeptide through proteolytic cleavage. The linker peptide containing the fusion peptide and linker sequence is extracted and removed, leaving only the desired mature peptide product without extra amino acids that would cause immunogenic responses.
Solution Approach 2:
The patent uses a linker peptide as an intermediary element that facilitates the expression and purification process. The linker peptide contains a proteolytic cleavage site that allows for the removal of the linker sequence after the peptide product is synthesized, enabling the transition from intermediate to final product.
2Productivity
If existing cleavage strategies using chemicals or proteases are used, then peptides are cleaved from the translated polypeptide, but extra amino acids remain in the final peptide product
Solution Approach 1:
The patent converts the potentially harmful effect of leaving extra amino acids into a beneficial process by designing the linker peptide to contain a specific proteolytic cleavage site. This allows the extra amino acids to be systematically removed through controlled proteolytic digestion, transforming a defect into a controlled purification step.
Solution Approach 2:
The patent changes the parameter of cleavage specificity by selecting a proteolytic enzyme with a specific recognition sequence (e.g., Arg-Arg-X-Leu) that precisely targets the linker peptide cleavage site. This ensures that cleavage occurs only at the desired location, removing the linker while preserving the intact peptide product.
3Adaptability or versatility
If recombinant DNA technology is used, then heterologous peptides can be produced using cellular machinery, but the process requires many complex biochemical processes including transcription, translation, protein folding, and post-translational modification
Solution Approach 1:
The patent applies segmentation by dividing the peptide production process into distinct functional modules: (1) expression of the recombinant polypeptide containing the fusion peptide and linker sequence, (2) proteolytic cleavage at the specific site, and (3) purification of the mature peptide. This segmentation simplifies the overall process by making each step independent and controllable.
Solution Approach 2:
The patent uses a cloned recombinant polypeptide sequence that copies the desired peptide structure within a larger expression framework. The fusion peptide and linker sequence are designed to be expressed together, allowing the cellular machinery to produce the intermediate form that can then be precisely processed into the final product.
4Productivity
If tandem repeats of desirable peptides are used to increase yield, then peptide production yield increases, but the chimeric protein must be designed so that cleavage sites are accessible to the cleaving agent
Solution Approach 1:
The patent applies local quality by creating a specific structural arrangement where the linker peptide and cleavage site are positioned in a localized region that is accessible to proteolytic enzymes. The linker peptide sequence and its spatial arrangement are optimized to ensure that the cleavage site is exposed and accessible, while the rest of the chimeric protein structure remains intact.
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
Produces peptides with exact target sequences, high yield, and minimal resource use, suitable for various applications, including medical and pharmaceutical uses, without extra amino acids or undesired modifications.
Implementation Method 1
enzyme-catalyzed and chemical proteolysis to cleave recombinant polypeptides with linked peptides, using proteases that recognize specific cleavage sites
Implementation Method 2
Proteolysis of the polypeptide yields the intermediate peptide, comprising basic amino acids at the carboxy-terminus of the target amino acid sequence of the product peptide
Implementation Method 3
chemical proteolysis to cleave the peptides from the translated polypeptide
Data Source
AI summary
This disclosure concerns production of product peptides with a target amino acid sequence by proteolysis of a recombinant polypeptide comprising specific protease recognition sites or chemical cleavage sequences. In some embodiments, the product peptide is released from repeating peptide units in the recombinant polypeptide by removal of intervening amino acid sequences by proteolysis by proteases that recognize sites within the intervening amino acid sequences and a carboxypeptidase, aminopeptidase, and/or further protease.