Antibody Expression Vectors with Chaperones for E. coli Periplasm
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Solution Overview
Problem
Accumulation of intact, properly assembled, active antibody fragments in Escherichia coli periplasm is challenging due to multiple steps including transcription, translation, non-natural amino acid incorporation, translocation, folding, and disulfide bond formation, with existing methods struggling to achieve high titer expression.
Innovation Solution
Utilization of engineered vectors and plasmids incorporating a partition B (parB) locus and chaperones like FkpA or Skp to facilitate proper folding and assembly of antibody fragments with site-specifically incorporated non-natural amino acids, optimizing expression through strategic nucleic acid sequences and orientations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional expression vectors are used in E. coli, then the system is simple and easy to manipulate, but the expression yield of antibody fragments with non-natural amino acids is extremely low
Solution Approach 1:
The patent combines multiple functional elements into a single integrated expression vector: the parB locus for plasmid stability, chaperone genes (FkpA, Skp, SurA) for protein folding assistance, and the antibody fragment expression cassette with non-natural amino acid incorporation capability. This merging of functions into one vector achieves high expression yield (up to 90-fold improvement) while maintaining practical usability through standardized modular components.
2Productivity
If multiple helper proteins are overexpressed to assist protein folding, then the titer of heterologous proteins increases, but the complexity of the expression system increases
Solution Approach 1:
The patent creates a universal expression vector design that can accommodate different antibody fragment sequences and non-natural amino acid incorporations while using a standardized set of helper proteins (parB locus, FkpA, Skp, SurA chaperones). This multi-functional vector platform has been successfully applied to express various difficult-to-express proteins, including different antibody fragments with site-specific non-natural amino acids, achieving high titers without requiring separate optimized systems for each protein.
3Reliability
If site-specific non-natural amino acid incorporation is implemented, then protein function and stability are improved, but the difficulty of achieving high expression titer increases significantly
Solution Approach 1:
The patent introduces chaperone proteins (FkpA, Skp, SurA) as intermediary molecules that facilitate the folding and assembly of antibody fragments containing non-natural amino acids. These chaperones act as mediators between the translated polypeptide chain and the final functional protein structure, helping to overcome the folding challenges posed by non-natural amino acids and enabling high expression titers of functional proteins with site-specific modifications.
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
Enhances the expression yield of antibody fragments by up to 90-fold or more, improving the titer and yield of recombinant proteins in E. coli systems.
Implementation Method 1
Chaperones bind to misfolded or unfolded proteins and facilitate proper folding. The present invention utilizes helper proteins and/or factors, including chaperones, in the design and engineering of vectors and plasmids therefrom to achieve optimization of titer of an antibody fragment
Implementation Method 2
a nucleic acid sequence comprising a partition B (parB) locus having the nucleobase sequence of SEQ ID NO: 12
Implementation Method 3
translocation across the inner membrane
Implementation Method 4
correct formation of intra- and inter-disulfide bonds
Data Source
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AI summary
Disclosed herein are methods, compositions and components for optimizing or increasing expression of a protein, polypeptide or fragment therefrom.