Recombinant C1q Protein Production via Subunit Segmentation
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Solution Overview
Problem
Current methods for producing the C1q protein are unsatisfactory due to its complex structure and the inability to produce complete, correctly structured protein through recombinant means, leading to reproducibility and biological safety concerns when extracted from human or animal serum.
Innovation Solution
Incorporating a peptide sequence DYKDDDDK at the C-terminal end of the C1qC subunit during recombinant production with C1qA and C1qB subunits in an in vitro cell culture, allowing for the generation of a C1q protein with a structure similar to its native form.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If C1q protein is extracted from human or animal serum, then complete C1q protein can be obtained, but biological safety is compromised due to contamination risks from viruses, prions, or parasites
Solution Approach 1:
The C1q protein is divided into three separate subunits (C1qA, C1qB, C1qC) that are expressed individually in recombinant cell cultures. Each subunit contains specific amino acid sequences that enable their separate production while maintaining the ability to self-assemble into the complete functional protein structure, thereby eliminating contamination risks from serum extraction.
Solution Approach 2:
A peptide sequence with at least 40% glutamic acid and/or aspartic acid residues serves as a mediator to facilitate the correct folding and assembly of recombinant C1q subunits. This peptide intermediary enables the recombinant subunits to form the native-like bouquet structure without requiring serum extraction, ensuring both safety and structural integrity.
2Reliability
If recombinant production of C1q subunits is attempted, then biological safety is improved, but complete and correctly structured C1q protein cannot be produced
Solution Approach 1:
Specific regions of the C1q subunits are engineered with enhanced properties: the C1qC subunit contains a peptide sequence with at least 40% glutamic acid and/or aspartic acid residues that locally facilitates correct folding and assembly. This local quality enhancement ensures that recombinant subunits can self-assemble into the complete native-like structure with proper disulfide bridges and collagen triple helices.
Solution Approach 2:
The amino acid composition of the C1qC subunit is modified by incorporating a peptide sequence with at least 40% glutamic acid and/or aspartic acid residues. This parameter change in charge distribution and chemical properties enables the recombinant subunits to achieve correct folding and assembly into the complete C1q structure, overcoming the limitation of previous recombinant approaches.
3Reliability
If C1q protein structure is maintained as native form, then functional activity is preserved, but production complexity increases due to multimeric assembly requirements
Solution Approach 1:
The recombinant C1q subunits are designed to self-assemble into the complete functional protein structure without requiring complex external assembly procedures. The C1qC subunit's peptide sequence with enhanced glutamic acid and/or aspartic acid content enables spontaneous correct folding and formation of disulfide bridges, allowing the subunits to automatically form the native-like bouquet structure with proper functional activity.
Data Source
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AI summary
The present invention relates to a method for recombinant production of a C1q protein or a variant of the C1q protein, in which the protein is recovered from an in vitro culture of cells expressing a C1qA subunit or a variant of the C1qA subunit, a C1qB subunit or a variant of the C1qB subunit, and a C1qC subunit or a variant of the C1qC subunit, in which at least one of the subunits or subunit variants also has at the N-terminus or C-terminus a sequence of amino acids of at least six residues, at least 40% of which are glutamic acid and/or aspartic acid residues.