Recombinant AIM Cysteine Substitution Prevents IgM Complex
Solution Overview
Problem
The challenge lies in producing recombinant AIM that maintains its function, avoids multimerization, and remains active by preventing complex formation with IgM pentamer, which is crucial for its pharmacological efficacy and stability during protein preparation and administration.
Innovation Solution
The approach involves substituting cysteine residues at specific positions, particularly amino acid number 168 and 277, with serine in the AIM sequence to prevent intermolecular disulfide bond formation, thereby preventing multimerization and complex formation with IgM pentamer, ensuring the recombinant AIM retains its biological activity and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If Escherichia coli is used as host cell for recombinant AIM production, then ease of handling and culturing is improved, but the protein forms incorrect higher-order structures due to preventing disulfide bond formation
Solution Approach 1:
The patent applies parameter changes by substituting cysteine residues with serine at specific positions (168 and/or 277) in the AIM amino acid sequence. This chemical modification prevents intermolecular disulfide bond formation while preserving intramolecular bonds, thereby preventing multimerization and maintaining proper monomeric higher-order structure of the recombinant protein in E. coli expression systems.
2Reliability
If mammalian cells are used as host cell for recombinant AIM production, then disulfide bond formation is enabled, but the recombinant protein may form multimers affecting purification and pharmacokinetics
Solution Approach 1:
The patent modifies the amino acid sequence by substituting cysteine with serine at positions 168 and/or 277 to control disulfide bond formation parameters. This selective substitution prevents intermolecular disulfide bonds that cause multimerization while allowing essential intramolecular disulfide bonds to form, thereby enabling proper protein folding without multimer formation in mammalian cell expression systems.
3Reliability
If wild-type recombinant AIM is produced, then biological activity is maintained, but the protein forms complexes with IgM pentamer causing inactivation
Solution Approach 1:
The patent applies parameter changes by substituting cysteine with serine at position 168, which is the specific residue responsible for forming disulfide bonds with IgM pentamer. This substitution prevents the harmful complex formation and inactivation while preserving the biological activity of AIM through maintenance of other essential cysteine residues and their intramolecular disulfide bonds.
4Productivity
If recombinant AIM is produced at high concentration, then productivity is improved, but the protein precipitates due to multimerization
Solution Approach 1:
The patent modifies the amino acid sequence parameters by substituting cysteine with serine at positions 168 and/or 277 to prevent intermolecular disulfide bond formation. This modification maintains protein solubility and prevents multimerization even at high production concentrations, thereby enabling high-productivity recombinant AIM production without precipitation.
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 enhances the recovery efficiency of recombinant AIM, prevents insolubilization, and maintains or improves its biological activity, allowing for effective administration without titer decrease due to inactivation by IgM pentamer complex formation.
Implementation Method 1
substituting cysteine residues at specific positions, particularly amino acid number 168 and 277, with serine in the AIM sequence to prevent intermolecular disulfide bond formation
Implementation Method 2
preventing multimerization and complex formation with IgM pentamer, ensuring the recombinant AIM retains its biological activity and stability
Data Source
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Figure 3
Figure 4~5
AI summary
The present invention relates to the provision of a recombinant AIM that maintains functions, does not multimerize, and is not inactivated by IgM (e.g., a protein containing an amino acid sequence wherein cysteine at amino acid number 168 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with another amino acid, and the like).