Proteolytic inactivation of select proteins in bacterial extracts for improved expression
Inventors
Thanos, Christopher D. • Murray, Christopher J. • Yang, Junhao • Stephenson, Heather
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11261219-B2
Publication Date
2022-03-01
Expiration Date
Abstract
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.
Core Innovation
The disclosed invention concerns modified Release Factor 1 (RF1) variants containing an OmpT1-cleavable bond in the switch-loop region. These modified RF1 variants retain stop-codon recognition and translation-termination activity during bacterial growth when OmpT is absent or uncleaved. Cleavage by OmpT1 reduces RF1 activity and competition at amber codons in cell-free synthesis extracts.
The system addresses the deleterious activity of RF1 competition at amber codons during in vitro cell-free protein synthesis. OmpT-mediated cleavage of RF1 variant A18 in cell-free extracts increases amber suppression and full-length IgG heavy-chain yield. This cleavage results in less truncated product compared with synthesis extracts containing uncleaved wild-type RF1.
Claims Coverage
The claims define three inventive features across independent claims covering a bacterial cell and cell-free synthesis methods.
Bacterial cell expressing OmpT1 and modified RF1
The claim covers a bacterial cell expressing OmpT1 and a modified RF1 that retains stop-codon recognition and translation-termination activity, wherein the modified RF1 includes an OmpT1-cleavable bond in the switch-loop region that is cleaved by OmpT1 expressed by the cell.
Reducing modified RF1 activity in cell-free synthesis
The claim covers reducing deleterious activity of modified RF1 in an in vitro cell-free synthesis system through an OmpT1-cleavable modified RF1, wherein the system produces a template-encoded protein of interest.
Reducing RF1 competition at an amber codon
The claim covers reducing RF1 competition at an amber codon in an in vitro cell-free synthesis system using a mutant RF1 containing an OmpT1-cleavable bond in the switch-loop region, in the presence of a template encoding a protein of interest with an amber codon.
Together, the independent claims cover OmpT1-cleavable modified RF1 in bacterial cells and in vitro cell-free synthesis systems to reduce RF1 activity or competition at an amber codon while producing a protein of interest.
Stated Advantages
OmpT-cleavable modified RF1 variants retain stop-codon recognition and translation-termination activity when OmpT is absent.
Cleavage of modified RF1 reduces RF1 activity and competition at amber codons in cell-free synthesis.
OmpT cleavage of RF1 variant A18 in cell-free extracts increases amber suppression and full-length IgG heavy-chain yield, with less truncated product than extracts containing uncleaved wild-type RF1.
Documented Applications
Cell-free synthesis of a protein of interest using an OmpT-cleavable modified RF1.
Cell-free production of IgG, including full-length IgG heavy-chain synthesis.
Placement and incorporation of a non-native amino acid at an amber codon in a protein of interest.
Interested in licensing this patent?