MEERKAT: MULTIPLEXED EFFICIENT EXPRESSION OF RECOMBINANT QconCATS
Lead Research Organisation:
University of Liverpool
Department Name: Institute of Integrative Biology
Abstract
Japan
People |
ORCID iD |
| Robert Beynon (Principal Investigator) |
Publications
Takemori A
(2020)
PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry
in Journal of Proteome Research
Takemori N
(2017)
MEERCAT: Multiplexed Efficient Cell Free Expression of Recombinant QconCATs For Large Scale Absolute Proteome Quantification.
in Molecular & cellular proteomics : MCP
| Description | We have shown that the production of designer proteins to allow quantification of proteins in a cell can be radically enhanced by leaving production in bacteria, by assembling the machines to make proteins in a testtubue in a cell-free system. This was a collaboration between colleagues in Japan and UK. The grant provided for travel to t Japan to bring the technology back to the UK |
| Exploitation Route | We have already proposed a 'human MEERCAT' project which would explore designer proteins to measure all human proteins. This is now under review for funding. |
| Sectors | Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology |
| Title | MEEERCAT |
| Description | A method to parallelise production of QconCAts, artificial designer proteins for proteome quantification,. |
| Type Of Material | Biological samples |
| Year Produced | 2017 |
| Provided To Others? | Yes |
| Impact | Too early although funding is being sought for a 'human MEERCAT' project |
| Description | Ehime University, japan |
| Organisation | Ehime University |
| Country | Japan |
| Sector | Academic/University |
| PI Contribution | Continued collaboration on an optimal MEERCAT system |
| Collaborator Contribution | This collaboration brings together two technologies that have the potential to produce a step change in the delivery of quantitative assessment of protein levels in biological systems. The first is QconCAT technology, invented by Beynon. QconCATs are artificial 'designer' proteins that are concatenations of peptides from different target proteins. When expressed in bacteria (E. coli) in the presence of stable isotope labelled amino acids, QconCATs generate multiplexed internal standards for quantification of up to 50 proteins at once. The biggest project to date (BBSRC funded, sLOLA, £3.9M) was the use of QconCATs to quantify 2,000 proteins in Saccharaomyces cerevisiae Each QconCAT gene must be designed, synthesised and expressed individually in bacteria and is thus not hugely efficient. However, Prof. Endo at Ehime University developed the second technology, cell free protein synthesis that is relevant to this proposal. In collaboration with Dr Takemori, we have demonstrated that a) QconCATs are efficiently synthesised in the cell-free system (CFS), b) all QconCATs that were toxic to E.coli are also efficiently expressed - about 10-12% of all QconCATs, c) QconCATs degraded in bacteria remain intact in CFS, d) multiple QconCATs can be co-expressed in a single CFS reaction, up to a plex level of at least 100. We refer to the multiplexed expression of QconCATs (themselves multiplexed standards) as MEERCAT. This is a major enhancement in QconCAT deployment. It is now feasible to contemplate the synthesis of a comprehensive panel of QconCATs to map, for example, the entire proteome of human or rice. However, the strategy and logistics of a 'human MEERCAT' or 'rice MEERCAT' project will take careful thought and design - this is the purpose of the collaboration. Page 11 / 1 9 To establish a 'human MEERCAT' project, we need to address several issues: the selection of optimal peptides as surrogate standards; the assembly of peptides into QconCATs, optimal gene design and synthesis, construction of a plasmid library containing the necessary sequence elements to ensure efficient transcription and translation of QconCATs. However, a complete MEERCAT project could require as many as 1000 QconCATs, which imposes considerable logistical problems in terms of organisation and delivery of subpanels of QconCATs to interested research groups. iverpool hosts GeneMill, a £3.8M BBSRC-funded Synthetic Biology 'Foundry'. Staffed with experienced postdoctoral researchers and state-of-the-art equipment, GeneMill has developed automated DNA fabrication, transformation, screening and fermentation platforms, which can be readily tailored to this project. After first designing the DNA constructs, GeneMill works closely with world-leading providers of short custom DNA and uses its robotics to rapidly fabricate 100s of full-length sequence-perfect clones per day. Furthermore, an integrated system of 'cherry- picking' and high-thoughput liquid handlers, plate reader, chillers and incubators allows complete automation of small-scale multiplexed in vitro protein expression. GeneMill has also implemented a fully-automated repository with a capacity to store and retrieve >120,000 samples. This will be invaluable to the longer-term goal of the 'Human MEERCAT' project - providing custom subpanels of QconCATs. |
| Impact | 1. One paper, in the top proteomics journal, , reported elsewhere. ONe grant proposal to develop the collaboration,. |
| Start Year | 2017 |