Systematic creation of fluorescent fusion polypeptides
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Publication Number
US-11702766-B2
Publication Date
2023-07-18
Expiration Date
Abstract
A method for creating a plasmid for use in producing a chimeric antibody, comprising (a) receiving a FAB region of the antibody; (b) receiving a fluorescent protein; (c) receiving a linker having length of at least 5 amino acids; (d) using the Gibson assembly process to join the FAB region, the fluorescent protein, and the linker into an expression plasmid.
Core Innovation
The invention describes a systematic method for creating fluorescent fusion antibody constructs (RFAB) for use in producing a chimeric antibody. The method receives a FAB region of the antibody, receives a fluorescent protein, and receives a linker having length of at least 5 amino acids (natural or unnatural, synthetic). The FAB region, fluorescent protein, and linker are combined to generate an expression plasmid for producing the chimeric antibody.
A key aspect is determining and selecting the linker length by comparison to a prototype “Durvasula” FAB/scFv length. The method compares lengths, including the Durvasula length and a length difference between the combined length of the FAB region and fluorescent protein and the Durvasula length. Depending on a threshold rule, the method either modifies the FAB region and uses a 5-amino-acid linker when the length difference is less than or equal to 5 amino acids, or uses a linker corresponding to the length difference when the length difference is greater than 5 amino acids.
The expression plasmid creation uses the Gibson assembly process to join the FAB region, the fluorescent protein, and the linker. The disclosure further describes preparing DNA segments for assembly, including PCR preparation of the FAB region, fluorescent protein, and linker components. The method also addresses linker elements and alignment to a template to determine added or removed linker sequences, aiming for optimized solubility and consistent expression of RFAB constructs.
Claims Coverage
The independent claim family provided includes one independent claim. The inventive features center on constructing an expression plasmid for producing a chimeric antibody by combining a FAB region and fluorescent protein via Gibson assembly using a linker of at least 5 amino acids, with dependent claim features adding linker-length selection logic relative to a “Durvasula length,” and additional limitations related to linker composition and segment preparation for assembly.
Gibson assembly of FAB, fluorescent protein, and at least 5-amino-acid linker into an expression plasmid
A method of creating a plasmid for use in producing a chimeric antibody by receiving a FAB region, receiving a fluorescent protein, receiving a linker having length of at least 5 amino acids (natural or unnatural, synthetic), and using the Gibson assembly process to join the FAB region, the fluorescent protein, and the linker into an expression plasmid.
Durvasula-length-based linker length difference threshold and conditional modification
Determining a difference in length between the FAB region plus fluorescent protein and the Durvasula length and, based on whether the difference is ≤5 amino acids or >5 amino acids, either modifying the FAB region and using a 5-amino-acid linker or using a linker equal to the difference.
Linker length selected based on comparison to Durvasula length
Receiving a linker by determining an amount based on whether the combined length of the FAB region and fluorescent protein is less than the Durvasula length, and receiving a linker whose length equals the determined amount.
PCR preparation of Gibson assembly components
Using PCR to produce volumes of the FAB region, the fluorescent protein, and the linker for use in the Gibson assembly process.
Linker comprising at least one natural amino acid
Providing a linker that comprises at least one natural amino acid.
Bacterial production using E. coli
Performing the method using bacteria comprising E. coli.
Across the independent claim and its dependent refinements, coverage is directed to plasmid construction for fluorescent fusion antibody constructs using Gibson assembly with a linker of at least 5 amino acids, and it further narrows linker length selection and related design logic using a “Durvasula length” comparison with threshold/conditional rules. Additional coverage includes PCR-based component preparation, linker composition requiring natural amino acids, and performing production using E. coli.
Stated Advantages
Optimized solubility and consistent expression of RFAB constructs.
Improved over prior restriction/ligation workflows with shorter turnaround and fewer cloning steps.
Documented Applications
Creating fluorescent fusion antibody constructs (RFAB) for producing a chimeric antibody by constructing an expression plasmid and performing production using bacteria comprising E. coli.
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