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Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry

[Image: see text] Directed Evolution is a key technology driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance l...

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Autores principales: Diefenbach, Xue W., Farasat, Iman, Guetschow, Erik D., Welch, Christopher J., Kennedy, Robert T., Sun, Shuwen, Moore, Jeffrey C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2018
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044804/
https://www.ncbi.nlm.nih.gov/pubmed/30023807
http://dx.doi.org/10.1021/acsomega.7b01973
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author Diefenbach, Xue W.
Farasat, Iman
Guetschow, Erik D.
Welch, Christopher J.
Kennedy, Robert T.
Sun, Shuwen
Moore, Jeffrey C.
author_facet Diefenbach, Xue W.
Farasat, Iman
Guetschow, Erik D.
Welch, Christopher J.
Kennedy, Robert T.
Sun, Shuwen
Moore, Jeffrey C.
author_sort Diefenbach, Xue W.
collection PubMed
description [Image: see text] Directed Evolution is a key technology driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance liquid chromatography-mass spectrometry (HPLC-MS), or optical detectors, which require either long cycle times or tailor-made substrates. To better fit modern, fast-paced process chemistry development where solutions are rapidly needed for new substrates, droplet microfluidics interfaced with electrospray ionization (ESI)-MS provides a label-free high-throughput screening platform. To apply this method to industrial enzyme screening and to explore potential approaches that may further improve the overall throughput, we optimized the existing droplet–MS methods. Carryover between droplets, traditionally a significant issue, was reduced to undetectable level by replacing the stainless steel ESI needle with a Teflon needle within a capillary electrophoresis (CE)–MS source. Throughput was improved to 3 Hz with a wide range of droplet sizes (10–50 nL) by tuning the sheath flow within the CE–MS source. The optimized method was demonstrated by screening reactions using two different transaminase libraries. Good correlations (r(2) ∼ 0.95) were found between the droplet–MS and LC–MS methods, with 100% match on hit variants. We further explored the capability of the system by performing in vitro transcription–translation inside the droplets and directly analyzing the intact reaction mixture droplets by MS. The synthesized protein attained comparable activity to the protein standard, and the complex samples appeared well tolerated by the MS. The success of the above applications indicates that the MS analysis of the microfluidic droplets is an available option for considerably accelerating the screening of enzyme evolution libraries.
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spelling pubmed-60448042018-07-16 Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry Diefenbach, Xue W. Farasat, Iman Guetschow, Erik D. Welch, Christopher J. Kennedy, Robert T. Sun, Shuwen Moore, Jeffrey C. ACS Omega [Image: see text] Directed Evolution is a key technology driving the utility of biocatalysis in pharmaceutical synthesis. Conventional approaches to Directed Evolution are conducted using bacterial cells expressing enzymes in microplates, with catalyzed reactions measured by HPLC, high-performance liquid chromatography-mass spectrometry (HPLC-MS), or optical detectors, which require either long cycle times or tailor-made substrates. To better fit modern, fast-paced process chemistry development where solutions are rapidly needed for new substrates, droplet microfluidics interfaced with electrospray ionization (ESI)-MS provides a label-free high-throughput screening platform. To apply this method to industrial enzyme screening and to explore potential approaches that may further improve the overall throughput, we optimized the existing droplet–MS methods. Carryover between droplets, traditionally a significant issue, was reduced to undetectable level by replacing the stainless steel ESI needle with a Teflon needle within a capillary electrophoresis (CE)–MS source. Throughput was improved to 3 Hz with a wide range of droplet sizes (10–50 nL) by tuning the sheath flow within the CE–MS source. The optimized method was demonstrated by screening reactions using two different transaminase libraries. Good correlations (r(2) ∼ 0.95) were found between the droplet–MS and LC–MS methods, with 100% match on hit variants. We further explored the capability of the system by performing in vitro transcription–translation inside the droplets and directly analyzing the intact reaction mixture droplets by MS. The synthesized protein attained comparable activity to the protein standard, and the complex samples appeared well tolerated by the MS. The success of the above applications indicates that the MS analysis of the microfluidic droplets is an available option for considerably accelerating the screening of enzyme evolution libraries. American Chemical Society 2018-02-05 /pmc/articles/PMC6044804/ /pubmed/30023807 http://dx.doi.org/10.1021/acsomega.7b01973 Text en Copyright © 2018 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
spellingShingle Diefenbach, Xue W.
Farasat, Iman
Guetschow, Erik D.
Welch, Christopher J.
Kennedy, Robert T.
Sun, Shuwen
Moore, Jeffrey C.
Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry
title Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry
title_full Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry
title_fullStr Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry
title_full_unstemmed Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry
title_short Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry
title_sort enabling biocatalysis by high-throughput protein engineering using droplet microfluidics coupled to mass spectrometry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6044804/
https://www.ncbi.nlm.nih.gov/pubmed/30023807
http://dx.doi.org/10.1021/acsomega.7b01973
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