Recombinant Der p1 Allergen Expression in Pichia pastoris
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Solution Overview
Problem
Current methods for treating dust mite allergies using crude allergen extracts are cumbersome, costly, and prone to contamination, leading to inconsistent quality and potential misdiagnosis, with long-term use causing adverse reactions due to complex composition and poor specificity.
Innovation Solution
Optimization of the proDer p1 gene for expression in the Pichia pastoris system, including codon optimization and an alpha-factor signal peptide, to enhance expression levels and purity of the recombinant Der p1 protein, followed by a scalable activation process and multi-step purification method, resulting in a recombinant protein with similar biological activity to the natural protein.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If crude extract of dust mite allergens is used for treatment, then allergic diseases can be treated, but the composition is complex leading to poor specificity and potential contamination
Solution Approach 1:
The patent extracts and isolates the specific Der p1 allergen component from the complex dust mite crude extract using recombinant expression technology. This allows obtaining a pure, single-component allergen product that maintains therapeutic efficacy while eliminating other complex components that cause poor specificity and contamination risks.
Solution Approach 2:
The patent creates a recombinant copy of the Der p1 allergen protein through genetic engineering in Pichia pastoris expression system. This synthetic copy has identical amino acid sequence to natural Der p1 but is produced in a controlled, pure form without contamination from other dust mite components, achieving both reliability and simplicity.
2Duration of action of stationary object
If crude extract is used for long-term treatment, then continuous therapy is possible, but adverse reactions occur due to complex composition
Solution Approach 1:
By extracting only the essential Der p1 component and removing all other components from the crude extract, the patent eliminates the source of adverse reactions while preserving the therapeutic allergenicity. This purified single-component formulation enables safe long-term treatment.
Solution Approach 2:
The patent applies local quality by providing a highly purified, homogeneous allergen product with consistent composition. This uniform quality ensures predictable immunological response and reduces variability in patient reactions, enabling safe long-term use.
3Measurement precision
If crude extract is used for diagnosis, then allergen response can be assessed, but specific determination of patient response to each component is impossible leading to misdiagnosis
Solution Approach 1:
The patent extracts the specific Der p1 component as a purified product, enabling diagnostic tests to specifically measure patient response to this single allergen component. This eliminates cross-reactivity issues and allows precise determination of component-specific IgE levels, preventing misdiagnosis.
4Productivity
If proDer p1 gene is expressed without optimization in Pichia pastoris system, then expression can occur, but the yield is low
Solution Approach 1:
The patent optimizes the proDer p1 gene sequence by modifying codon usage to match the preferences of Pichia pastoris, and by optimizing expression parameters such as induction conditions and cultivation parameters. These parameter changes dramatically increase the expression yield of Der p1 protein while maintaining the simplicity of the overall manufacturing process.
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
The optimized recombinant Der p1 protein achieves higher expression levels and improved biological activity, reducing adverse reactions and enhancing the specificity and safety of immunotherapy, while maintaining effective allergenicity for diagnostic and therapeutic applications.
Implementation Method 1
optimize the proDer p1 gene in the Pichia pastoris expression system, and add an acting element to increase the expression of proDer p1 in molecular level
Implementation Method 2
The instant application provides a DNA sequence encoding proDer p1 protein, having a base sequence as shown in SEQ ID NO: 1. This sequence has been codon-optimized for the Pichia pastoris expression system
Implementation Method 3
add an acting element to increase the expression of proDer p1 in molecular level
Implementation Method 4
multi-step purification method, resulting in a recombinant protein with similar biological activity to the natural protein
Implementation Method 5
the activation process of proDer p1 was further studied and optimized by the inventors, in which a more operational and scalable activation process was adopted
Data Source
AI summary
A DNA sequence encoding Der p1 protein having a particular base sequence is codon-optimized for the Pichia pastoris expression system, which is conducive to expressing Der p1 in Pichia pastoris. After gene optimization and adding an activating element to increase the expression of Der p1 in molecular level, it was found that Der p1 is expressed at a higher level as compared with the prior art and has biological activity similar to the natural protein.