In Vitro RNA Display for scFv Antibody Expression
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Solution Overview
Problem
Current in vitro RNA display systems are inefficient in expressing multi-domain antibodies like single chain antibody (scFv) molecules due to reaction conditions that prevent the formation of intra-chain disulphide bonds, leading to incorrect folding and loss of full-length scFv cDNA during PCR amplification.
Innovation Solution
The method involves crosslinking scFv mRNA with a puromycin or analogue and a nucleic acid linker, translating it under mildly oxidizing conditions with protein disulphide isomerase, and using affinity-based magnetic beads for selection and purification, allowing for the reliable expression and selection of scFv molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current in vitro RNA display systems are used to express scFv molecules, then single antibody variable domains can be expressed, but multi-domain antibodies like scFv molecules cannot be efficiently expressed due to loss of full length scFv cDNA during PCR amplification
Solution Approach 1:
The invention changes the chemical environment parameters by using mildly oxidizing conditions instead of reducing conditions, and by using PDI (protein disulphide isomerase) to catalyze disulphide bond formation. This resolves the contradiction by enabling correct scFv folding while maintaining cDNA integrity during PCR amplification.
Solution Approach 2:
The invention introduces PDI (protein disulphide isomerase) as an intermediary enzyme that facilitates disulphide bond formation in scFv molecules. This mediator enables correct folding of multi-domain antibodies without compromising cDNA stability during subsequent PCR amplification steps.
2Productivity
If reducing conditions are used in in vitro translation, then protein expression can proceed, but intra-chain disulphide bonds cannot form leading to incorrect folding
Solution Approach 1:
The invention changes the oxidation state parameter from reducing to mildly oxidizing conditions, enabling disulphide bond formation while maintaining adequate protein expression levels through the catalytic action of PDI.
Solution Approach 2:
The invention uses mildly oxidizing conditions to accelerate disulphide bond formation in scFv molecules, enabling correct folding while maintaining protein expression through PDI catalysis.
3Quantity of substance
If repeated PCR amplification is performed on scFv cDNA, then library amplification can be achieved, but full length scFv cDNA is lost through the amplification process
Solution Approach 1:
The invention changes the translation conditions to mildly oxidizing environment with PDI, which produces correctly folded scFv proteins that maintain associated cDNA integrity during PCR amplification, reducing cDNA loss while achieving library amplification.
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 approach enables simpler, faster, and more efficient in vitro RNA display for scFv molecules, enhancing their functional expression and selection while maintaining correct folding, and can be applied to all classes of proteins.
Implementation Method 1
providing a puromycin or analogue thereof crosslinked scFv mRNA molecule, said molecule comprising an mRNA encoding a 5′ scFv and a 3′ spacer sequence, which molecule is crosslinked to a single stranded nucleic acid linker
Implementation Method 2
the linker comprising a puromycin, or analogue thereof, at a 3′ end and a Psoralen C6 at the 5′ end
Implementation Method 3
in the presence of oxidized glutathione/reduced glutathione and PDI (protein disulphide isomerase) under conditions such that a labeled puromycin-crosslinked scFv mRNA/protein molecule is formed
Implementation Method 4
PDI (protein disulphide isomerase)
Implementation Method 5
recovering the purified labeled puromycin-crosslinked scFv mRNA/protein molecules using affinity based magnetic beads
Data Source
AI summary
The present invention features improved methods of in vitro RNA display to allow reliable expression and selection of scFv antibody molecules from expression libraries. The improved methods, in part, involve the use of mildly reducing conditions, which favor of scFv intra-chain disulphide bond and thus correct folding of the scFv antibody molecules. Although particularly suited to expression and selection of scFv antibody molecules, the methods of the invention are also expedient for in vitro RNA display of all classes of protein.