RF1 Proteolytic Cleavage for Non-Natural Amino Acid Yield
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Solution Overview
Problem
In bacterial cell-free extracts, the efficiency of protein synthesis is hindered by native proteins that inhibit translation, such as Release Factor 1 (RF1), which recognizes the amber stop codon and promotes premature truncation of proteins incorporating non-native amino acids, reducing yield.
Innovation Solution
Introduction of Outer Membrane Protein T1 (OmpT1) protease cleavage sites into RF1, allowing for proteolytic inactivation upon cell lysis, thereby reducing RF1 activity and increasing the incorporation of non-native amino acids into proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If RF1 is present in the cell-free extract, then translation termination is maintained, but the yield of full-length proteins with non-native amino acids is reduced due to premature truncation
Solution Approach 1:
The patent extracts and removes RF1 from the cell-free extract through proteolytic cleavage. By introducing a protease cleavage site into RF1 and using a protease (such as protease K) to cleave this site, RF1 is degraded and removed from the extract, eliminating its harmful termination activity while preserving the desired protein synthesis function.
Solution Approach 2:
The patent converts the harmful RF1 protein into a beneficial outcome by introducing a protease cleavage site. The cleavage site is designed to be inaccessible during cell growth (maintaining RF1 function) but becomes accessible after cell lysis, allowing controlled degradation of RF1. This transforms RF1 from a harmful factor into a controlled element that can be removed at the desired time.
2Productivity
If proteolytic cleavage sites are introduced into RF1, then RF1 activity is reduced and protein yield increases, but the complexity of the extract preparation increases
Solution Approach 1:
The patent performs preliminary action by introducing the protease cleavage site into RF1 during the strain construction phase. This preliminary modification allows the cleavage to occur automatically during normal cell growth without requiring additional steps during extract preparation. The cleavage site is designed to be activated by standard protease treatment that is already part of the extract preparation protocol.
Solution Approach 2:
The patent implements self-service by designing the cleavage site to be automatically activated during the standard cell lysis and extract preparation process. The protease cleavage occurs as part of the routine protocol without requiring separate or additional steps, making the system self-regulating and simplifying the overall process despite the added complexity of strain construction.
3Object-generated harmful factors
If the protease cleavage site is made accessible during cell growth, then RF1 can be inactivated, but essential bacterial proteins are degraded
Solution Approach 1:
The patent applies local quality by making the cleavage site accessible only in specific locations and conditions. The cleavage site is positioned in a region of RF1 that becomes accessible to proteases after cell lysis, while remaining protected during cell growth. This localized accessibility ensures that only RF1 is degraded, not essential bacterial proteins, as the cleavage site is not exposed to proteases in the intact cell.
Solution Approach 2:
The patent implements dynamics by designing the cleavage site accessibility to change over time. The site is inaccessible during cell growth (when proteases are not active or are sequestered) but becomes accessible after cell lysis when proteases are released and activated. This dynamic accessibility allows temporal control of RF1 inactivation without affecting essential proteins during growth.
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 enhances the yield of full-length proteins with non-native amino acids by reducing RF1 activity, leading to increased incorporation of these amino acids at defined positions, improving protein synthesis efficiency in cell-free systems.
Implementation Method 1
The invention provides methods for reducing the deleterious activity of a modified essential target protein in an in vitro cell free synthesis system by inactivating the target protein with an OmpT1 protease. The modified target protein contains a scissile OmpT1 peptide bond that is cleaved by the protease.
Implementation Method 2
combining a nucleic acid template with a bacterial extract sufficient to translate the nucleic acid template; and expressing a protein from the nucleic acid template
Data Source
AI summary
The present disclosure provides modified proteins that are capable of being cleaved by the protease OmpT1. The proteins can be modified in an exposed surface motif to incorporate OmpT1 cleavage sites. Also provided are nucleic acids encoding the modified proteins, bacterial cells that express the modified proteins, and cell free synthesis systems containing modified RF1. The disclosure further provides methods for reducing the deleterious activity of a modified protein in a cell free synthesis system by contacting the modified protein with OmpT1. Also provided are methods for reducing RF1 competition at an amber codon in the cell free synthesis system, and methods for expressing a protein in the cell free synthesis system. The modified proteins of the invention can be used to increase the yield of proteins having non-natural amino acids incorporated at an amber codon.