Recombinant CD74 Polypeptides Bacterial Expression
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Solution Overview
Problem
There is a lack of commercially available soluble recombinant CD74 (invariant chain) polypeptides capable of binding CD74 ligands such as MIF, D-DT, RTL1000, and DRα1, which are essential for research purposes.
Innovation Solution
Development of soluble recombinant CD74 polypeptides with specific sequences (SEQ ID NO: 1, 3, 5) or their conservative amino acid substitution mutants that maintain at least 50% binding affinity to CD74 ligands, along with corresponding codon-optimized polynucleotides and expression vectors for bacterial expression, allowing for the production and purification of these polypeptides in E. coli cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If soluble recombinant CD74 polypeptides are produced in bacterial cells, then commercial availability and research accessibility are improved, but the complexity of ensuring proper folding and binding affinity is increased
Solution Approach 1:
The CD74 polypeptide is divided into two domains: an N-terminal unfolded domain and a C-terminal organized domain. This segmentation allows the C-terminal domain to independently trimerize and bind ligands while the N-terminal domain remains flexible, simplifying the overall folding requirements in bacterial expression systems.
Solution Approach 2:
The patent employs conservative amino acid substitution mutations to modify the CD74 sequence while maintaining at least 50% binding affinity. This parameter change approach allows optimization of expression and folding properties in bacterial cells without losing the essential ligand-binding function.
2Productivity
If conservative amino acid substitution mutations are introduced to optimize expression, then expression levels are improved, but maintaining binding affinity becomes more challenging
Solution Approach 1:
The patent systematically applies conservative amino acid substitutions that maintain similar chemical properties (e.g., hydrophobic, charged, polar residues). This controlled parameter change allows optimization of expression levels while the conservative nature of substitutions preserves the binding interface characteristics, ensuring at least 50% affinity retention.
Solution Approach 2:
The patent creates multiple variant polypeptides with different conservative mutations, effectively generating a library of 'disposable' variants that can be screened. This allows identification of variants with optimal expression levels while maintaining sufficient binding affinity for research applications.
Data Source
AI summary
Disclosed are recombinant CD74 polypeptides mutated relative to the naturally occurring CD74 polypeptides with improved properties such as binding of CD74 ligands such as MIF and RTL1000 as well as polynucleotides that encode the polypeptides, expression vectors comprising the polynucleotides, bacteria that include the expression vectors, and methods of making the recombinant polypeptides.