Cell-Free Protein Synthesis with Chaperones
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Solution Overview
Problem
Bacterial cell-free protein synthesis systems face challenges with slower growth rates and lower protein synthetic activity due to overexpression of proteins, leading to improper folding and loss of biological activity, necessitating improved extracts with enhanced chaperone expression for proper protein folding.
Innovation Solution
Incorporating a bacterial extract with an active oxidative phosphorylation system and exogenous protein chaperones like disulfide isomerase and peptidyl-prolyl cis-trans isomerase, expressed at high concentrations, to improve the expression and folding of biologically active proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If proteins are overexpressed during bacterial growth to provide cell-free synthesis systems with altered properties, then the protein synthetic activity in extracts is improved, but the bacterial growth rate slows down and the protein folding quality deteriorates
Solution Approach 1:
The patent applies preliminary action by pre-expressing chaperone proteins (DsbC and FkpA) in the bacterial host before preparing the cell-free extract. This ensures that chaperones are already present in the extract at high concentrations, enabling proper protein folding to occur during the cell-free synthesis process without requiring additional optimization steps later.
Solution Approach 2:
The patent uses chaperone proteins as intermediaries to mediate between the overexpressed target protein and the folding environment. Specifically, DsbC acts as an intermediary for disulfide bond formation and FkpA for proline isomerization, facilitating proper folding of the target protein during cell-free synthesis.
2Reliability
If chaperone proteins are added to the cell-free synthesis system to improve proper folding, then the biological activity of expressed proteins is improved, but the system complexity increases
Solution Approach 1:
The patent applies self-service by engineering the bacterial host to autonomously produce and accumulate chaperone proteins (DsbC and FkpA) during growth. The cell-free extract then inherently contains these chaperones, eliminating the need for external addition or complex optimization of chaperone concentrations during the cell-free synthesis process.
Solution Approach 2:
The patent changes the concentration parameter of chaperone proteins in the cell-free extract by engineering the bacterial host to overexpress them. This results in extracts with inherently high chaperone concentrations, simplifying the system by eliminating the need for external chaperone addition and optimization.
3Productivity
If extracts are prepared from bacteria overexpressing chaperones to synthesize large amounts of properly folded protein, then the yield of biologically active protein is improved, but the extraction process complexity increases
Solution Approach 1:
The bacterial host autonomously produces and accumulates chaperone proteins during growth, and these chaperones are automatically incorporated into the cell-free extract during preparation. This self-service approach eliminates the need for complex post-extraction chaperone addition or optimization steps.
Solution Approach 2:
The patent applies preliminary action by pre-expressing chaperone proteins in the bacterial host before extract preparation. This ensures that chaperones are already present in the extract at high concentrations, enabling proper protein folding to occur during the cell-free synthesis process without requiring additional optimization steps later.
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
The method significantly increases the production of properly folded and biologically active proteins, achieving higher yields and maintaining bacterial growth rates by synergistically enhancing protein expression and folding processes.
Implementation Method 1
The cell-free synthesis system comprises a bacterial extract having an active oxidative phosphorylation system
Implementation Method 2
exogenous protein disulfide isomerase
Implementation Method 3
exogenous peptidyl-prolyl cis/trans isomerase
Data Source
Figure 1
Figure 2A
Figure 2B
AI summary
The present disclosure describes methods and systems for improving the expression of a properly folded, biologically active protein of interest in a cell free synthesis system. The methods and systems use a bacterial cell free extract having an active oxidative phosphorylation system, and include an exogenous protein chaperone. The exogenous protein chaperone can be expressed by the bacteria used to prepare the cell free extract. The exogenous protein chaperone can be a protein disulfide isomerase and/or a peptidyl-prolyl cis-trans isomerase. The inventors discovered that the combination of a protein disulfide isomerase and a peptidyl-prolyl cis-trans isomerase produces a synergistic increase in the amount of properly folded, biologically active protein of interest.