Engineering Cell-free Systems by Chemoproteomic-assisted Phenotypic Screening

Overview

Journal RSC Chem Biol

Publisher Royal Society of Chemistry

Specialty Biology

Date 2024 Apr 5

PMID 38576719

Authors

Zarina Levitskaya

Zheng Ser

Hiromi Koh

Wang Shi Mei

Sharon Chee

Radoslaw Mikolaj Sobota

John F Ghadessy

Affiliations

Soon will be listed here.

Abstract

Phenotypic screening is a valuable tool to both understand and engineer complex biological systems. We demonstrate the functionality of this approach in the development of cell-free protein synthesis (CFPS) technology. Phenotypic screening identified numerous compounds that enhanced protein production in yeast lysate CFPS reactions. Notably, many of these were competitive ATP kinase inhibitors, with the exploitation of their inherent substrate promiscuity redirecting ATP flux towards heterologous protein expression. Chemoproteomic-guided strain engineering partially phenocopied drug effects, with a 30% increase in protein yield observed upon deletion of the ATP-consuming SSA1 component of the HSP70 chaperone. Moreover, drug-mediated metabolic rewiring coupled with template optimization generated the highest protein yields in yeast CFPS to date using a hitherto less efficient, but more cost-effective glucose energy regeneration system. Our approach highlights the utility of target-agnostic phenotypic screening and target identification to deconvolute cell-lysate complexity, adding to the expanding repertoire of strategies for improving CFPS.

References

Schoborg J, Hodgman C, Anderson M, Jewett M . Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis. Biotechnol J. 2013; 9(5):630-40. DOI: 10.1002/biot.201300383. View

Franken H, Mathieson T, Childs D, Sweetman G, Werner T, Togel I . Thermal proteome profiling for unbiased identification of direct and indirect drug targets using multiplexed quantitative mass spectrometry. Nat Protoc. 2015; 10(10):1567-93. DOI: 10.1038/nprot.2015.101. View

Martin R, Des Soye B, Kwon Y, Kay J, Davis R, Thomas P . Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids. Nat Commun. 2018; 9(1):1203. PMC: 5865108. DOI: 10.1038/s41467-018-03469-5. View

Martin R, Majewska N, Chen C, Albanetti T, Jimenez R, Schmelzer A . Development of a CHO-Based Cell-Free Platform for Synthesis of Active Monoclonal Antibodies. ACS Synth Biol. 2017; 6(7):1370-1379. DOI: 10.1021/acssynbio.7b00001. View

Tay Y, Ho C, Droge P, Ghadessy F . Selection of bacteriophage lambda integrases with altered recombination specificity by in vitro compartmentalization. Nucleic Acids Res. 2009; 38(4):e25. PMC: 2831311. DOI: 10.1093/nar/gkp1089. View

Wuu J, Swartz J . High yield cell-free production of integral membrane proteins without refolding or detergents. Biochim Biophys Acta. 2008; 1778(5):1237-50. DOI: 10.1016/j.bbamem.2008.01.023. View

Wang P, Fujishima K, Berhanu S, Kuruma Y, Jia T, Khusnutdinova A . A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis. ACS Synth Biol. 2019; 9(1):36-42. DOI: 10.1021/acssynbio.9b00456. View

Remsing Rix L, Rix U, Colinge J, Hantschel O, Bennett K, Stranzl T . Global target profile of the kinase inhibitor bosutinib in primary chronic myeloid leukemia cells. Leukemia. 2008; 23(3):477-85. DOI: 10.1038/leu.2008.334. View

Werner-Washburne M, Becker J, Craig E . Yeast Hsp70 RNA levels vary in response to the physiological status of the cell. J Bacteriol. 1989; 171(5):2680-8. PMC: 209952. DOI: 10.1128/jb.171.5.2680-2688.1989. View

10.

Burrington L, Watts K, Oza J . Characterizing and Improving Reaction Times for Based Cell-Free Protein Synthesis. ACS Synth Biol. 2021; 10(8):1821-1829. DOI: 10.1021/acssynbio.1c00195. View

11.

Silverman A, Karim A, Jewett M . Cell-free gene expression: an expanded repertoire of applications. Nat Rev Genet. 2019; 21(3):151-170. DOI: 10.1038/s41576-019-0186-3. View

12.

Thomas P, Ebert D, Muruganujan A, Mushayahama T, Albou L, Mi H . PANTHER: Making genome-scale phylogenetics accessible to all. Protein Sci. 2021; 31(1):8-22. PMC: 8740835. DOI: 10.1002/pro.4218. View

13.

Caschera F, Noireaux V . Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system. Biochimie. 2013; 99:162-8. DOI: 10.1016/j.biochi.2013.11.025. View

14.

Kim T, Keum J, Oh I, Choi C, Kim H, Kim D . An economical and highly productive cell-free protein synthesis system utilizing fructose-1,6-bisphosphate as an energy source. J Biotechnol. 2007; 130(4):389-93. DOI: 10.1016/j.jbiotec.2007.05.002. View

15.

Tabuchi T, Yokobayashi Y . Cell-free riboswitches. RSC Chem Biol. 2021; 2(5):1430-1440. PMC: 8496063. DOI: 10.1039/d1cb00138h. View

16.

Panthu B, Ohlmann T, Perrier J, Schlattner U, Jalinot P, Elena-Herrmann B . Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems. ACS Synth Biol. 2017; 7(1):218-226. DOI: 10.1021/acssynbio.7b00280. View

17.

Jiang X, Ookubo Y, Fujii I, Nakano H, Yamane T . Expression of Fab fragment of catalytic antibody 6D9 in an Escherichia coli in vitro coupled transcription/translation system. FEBS Lett. 2002; 514(2-3):290-4. DOI: 10.1016/s0014-5793(02)02383-9. View

18.

Goh W, Lane D, Ghadessy F . Development of a novel multiplex in vitro binding assay to profile p53-DNA interactions. Cell Cycle. 2010; 9(15):3030-8. DOI: 10.4161/cc.9.15.12436. View

19.

Doncheva N, Morris J, Gorodkin J, Jensen L . Cytoscape StringApp: Network Analysis and Visualization of Proteomics Data. J Proteome Res. 2018; 18(2):623-632. PMC: 6800166. DOI: 10.1021/acs.jproteome.8b00702. View

20.

Maini R, Umemoto S, Suga H . Ribosome-mediated synthesis of natural product-like peptides via cell-free translation. Curr Opin Chem Biol. 2016; 34:44-52. DOI: 10.1016/j.cbpa.2016.06.006. View