Ribosome Display Complex Formation for Antibody Multimers
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Solution Overview
Problem
In vitro selection systems face challenges in efficiently displaying protein multimers, such as antibodies, due to decreased translation efficiency and display efficiency when translating mRNAs of H chain and L chain simultaneously, which affects the formation of ribosome display complexes.
Innovation Solution
The method involves forming a ribosome display complex with the L chain mRNA by adding a spacer gene and removing the stop codon, followed by translating the H chain mRNA separately and mixing the reaction products to associate the H chain with the L chain, thereby enhancing the efficiency of forming the ribosome display complex.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If mRNAs of H chain and L chain are simultaneously translated in the same in vitro translation system, then both chains are produced in the system, but the translation efficiency of H chain decreases and the display efficiency of L chain on ribosome decreases, markedly decreasing the formation efficiency of ribosome display complex
Solution Approach 1:
The patent divides the translation process into two separate reactions: Reaction 1 translates only the L chain mRNA to produce L chain protein displayed on ribosomes, while Reaction 2 translates only the H chain mRNA to produce free H chain protein. This segmentation eliminates competitive inhibition between the two mRNAs for translation resources, allowing each chain to be translated with high efficiency in its dedicated reaction system.
Solution Approach 2:
The patent extracts the H chain translation from the L chain translation system. By removing the H chain mRNA from Reaction 1 and translating it separately in Reaction 2, the system eliminates the harmful competition for translation factors and ribosomes, thereby resolving the contradiction between producing both chains and maintaining high translation efficiency.
2Reliability
If treatments are performed to keep ribosome on mRNA (such as removal of stop codon, addition of arrest sequence, removal of release factor or ribosome recycling factor), then L chain display efficiency on ribosome is maintained, but translation efficiency of H chain decreases when translated in the same system
Solution Approach 1:
The patent segments the translation reactions so that L chain translation (Reaction 1) occurs in a system with modified conditions (removal of stop codon, addition of arrest sequence) that ensure ribosome retention and high display efficiency, while H chain translation (Reaction 2) occurs in a separate system with normal translation factors that ensures high translation efficiency. Each reaction is optimized for its specific purpose without compromising the other.
Data Source
AI summary
The invention provides a method of producing a protein multimer-nucleic acid complex comprising a protein multimer and any one target component of the protein multimer. A nucleic acid encoding the target component is subjected to in vitro translation to provide a translation product containing a target component-nucleic acid complex of the target component and the nucleic acid encoding the target component. A nucleic acid encoding a non-target component constituting the protein multimer together with the target component is translated into the non-target component by adding the nucleic acid encoding the non-target component to the previously provided translation product to provide the non-target component. The non-target component then is associated with the target component contained in the target component-nucleic acid complex to form a protein multimer, thus affording the protein multimer-nucleic acid complex of the protein multimer and the nucleic acid encoding the target component.