Truncated Protein A Polypeptides for Stable E. coli Expression
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Solution Overview
Problem
Current methods for producing protein A polypeptides require pathogenic Staphylococcus aureus hosts, which are hazardous and inefficient, as they are prone to degradation and toxicity issues, limiting their use in non-pathogenic bacteria like E. coli.
Innovation Solution
Development of novel nucleic acid sequences encoding truncated Staphylococcus aureus protein A polypeptides that include a portion of the X-domain, lack a signal sequence, and bind specifically to the Fc region of IgG immunoglobulin, enabling efficient expression and immobilization in non-pathogenic bacteria like E. coli without significant degradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If full-length protein A is expressed in non-pathogenic bacteria like E. coli, then production safety is improved, but the protein is significantly degraded and expression efficiency deteriorates
Solution Approach 1:
The protein A molecule is divided into functional domains (IgG-binding domains E, D, A, B, C and X-domain). The invention selectively combines specific domains (at least one IgG-binding domain and a portion of the X-domain) to create truncated versions that maintain function while improving stability in non-pathogenic hosts.
Solution Approach 2:
The signal sequence is removed from the protein A construct. The patent specifically describes nucleic acids encoding truncated protein A that do not include a signal sequence, preventing mislocalization and degradation while maintaining the IgG-binding function in the cytoplasm of E. coli.
2Reliability
If truncated protein A lacking X-domain is produced, then expression stability in E. coli is improved, but immobilization capability for affinity chromatography deteriorates
Solution Approach 1:
The invention applies local quality by selectively including a portion of the X-domain (without the complete X-domain or signal sequence) to provide specific functional properties. The truncated protein A maintains the IgG-binding domains for function while including enough X-domain sequence to enable immobilization on solid supports for affinity chromatography.
3Reliability
If complete native protein A sequence is used, then natural function is preserved, but expression in heterologous hosts becomes difficult and hazardous
Solution Approach 1:
The nucleic acid sequences are codon-optimized for expression in non-pathogenic hosts like E. coli. The patent describes nucleic acids that are codon-optimized to enable efficient expression in heterologous hosts while encoding truncated protein A polypeptides that retain IgG-binding function.
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 truncated protein A polypeptides are stably expressed in E. coli, maintaining the X-domain's immobilization properties, enhancing their use in affinity chromatography and reducing the risks associated with pathogenic hosts, while achieving comparable binding capacities to commercial products.
Implementation Method 1
binds specifically to an Fc region of an IgG immunoglobulin
Implementation Method 2
immobilized for use as an affinity chromatography reagent
Data Source
AI summary
Disclosed are new recombinant nucleic acids encoding protein A polypeptides and methods of using these nucleic acids.