Ancestral Sequence Reconstruction for Stable Spike Protein Production
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Solution Overview
Problem
The production of stable and high-titer viral proteins, such as the spike protein of coronaviruses, is challenging due to low stability and low expression levels, which hampers the development of low-cost vaccines that can be administered at room temperature.
Innovation Solution
The use of ancestral sequence reconstruction to generate stable coronavirus spike proteins with robust folds, which are produced through a method involving the determination of ancestral protein sequences at branch points in phylogenetic trees, followed by the replacement of specific domains with corresponding domains from homologous sequences, resulting in proteins suitable for use as antigens and vaccine candidates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If ancestral sequence reconstruction is used to produce spike proteins, then stability and expression titer are improved, but the complexity of the method increases compared to traditional approaches
Solution Approach 1:
The patent applies preliminary action by reconstructing ancestral sequences before protein expression to inherently stabilize the protein structure. The method determines ancestral amino acid sequences at branch points in phylogenetic trees, then produces proteins based on these pre-determined sequences, eliminating the need for subsequent stabilization interventions.
Solution Approach 2:
The patent changes the fundamental parameter of the protein sequence by using ancestral sequences instead of contemporary sequences. This parameter change at the amino acid level fundamentally alters the protein's stability properties and expression characteristics, achieving high stability and high titer simultaneously.
2Productivity
If ancestral sequence reconstruction is used to produce spike proteins, then expression titer is improved, but the time required for sequence determination and testing increases
Solution Approach 1:
The method performs preliminary determination of ancestral sequences through phylogenetic analysis before expression testing. By pre-determining the optimal ancestral sequence at a branch point, the method reduces the number of iterative testing rounds needed, as the ancestral sequence inherently provides the desired high expression titer and stability.
Solution Approach 2:
The patent uses computational copying of ancestral sequence information from phylogenetic data to generate the protein sequence. Instead of extensive experimental trial-and-error, the method copies the proven ancestral sequence pattern that naturally confers high expression and stability properties.
3Reliability
If domain replacement with homologous sequences is performed, then protein stability is improved, but the precision of sequence alignment and replacement increases
Solution Approach 1:
The patent applies local quality by performing domain replacement only in specific regions of the spike protein where homologous sequences are inserted. This localized modification approach maintains the overall protein structure while improving stability in specific domains, reducing the precision burden compared to complete sequence redesign.
Solution Approach 2:
The patent creates a composite protein structure by combining ancestral sequence regions with homologous domain sequences. This composite approach leverages the stability of ancestral sequences in certain regions while incorporating functional domains from homologous proteins, achieving enhanced overall stability through strategic composition.
Data Source
AI summary
A protein, such as an antigenic protein, is produced by determining an amino acid sequence of an ancestral version of a given protein in an ancestral sequence reconstruction method based on a plurality of homologous amino acid sequences of the given protein. A domain of the amino acid sequence of the ancestral version of the given protein is replaced with a corresponding domain derived from an amino acid sequence of the given protein or a homologous version thereof. The protein thereby comprises the amino acid sequence obtained by replacing the domain of the amino acid sequence of the ancestral version of the given protein with the corresponding domain derived from the amino acid sequence of the given protein or the homologous version thereof. The protein is suitable as antigen, as vaccine candidate and/or for structural studies.