Assessment of recombinant protein production in E. coli with Time-Gated Surface Enhanced Raman Spectroscopy (TG-SERS)

Martin Kögler; Jaakko Itkonen; Tapani Viitala; Marco G. Casteleijn

doi:10.1038/s41598-020-59091-3

Assessment of recombinant protein production in E. coli with Time-Gated Surface Enhanced Raman Spectroscopy (TG-SERS)

Martin Kögler, Jaakko Itkonen, Tapani Viitala, Marco G. Casteleijn^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Scientific › peer-review

18 Citations (Scopus)

Abstract

Time-Gated Surface-Enhanced Raman spectroscopy (TG-SERS) was utilized to assess recombinant protein production in Escherichia coli. TG-SERS suppressed the fluorescence signal from the biomolecules in the bacteria and the culture media. Characteristic protein signatures at different time points of the cell cultivation were observed and compared to conventional continuous wave (CW)-Raman with SERS. TG-SERS can distinguish discrete features of proteins such as the secondary structures and is therefore indicative of folding or unfolding of the protein. A novel method utilizing nanofibrillar cellulose as a stabilizing agent for nanoparticles and bacterial cells was used for the first time in order to boost the Raman signal, while simultaneously suppressing background signals. We evaluated the expression of hCNTF, hHspA1, and hHsp27 in complex media using the batch fermentation mode. HCNTF was also cultivated using EnBase in a fed-batch like mode. HspA1 expressed poorly due to aggregation problems within the cell, while hCNTF expressed in batch mode was correctly folded and protein instabilities were identified in the EnBase cultivation. Time-gated Raman spectroscopy showed to be a powerful tool to evaluate protein production and correct folding within living E. coli cells during the cultivation.

Original language	English
Article number	2472
Journal	Scientific Reports
Volume	10
Issue number	1
DOIs	https://doi.org/10.1038/s41598-020-59091-3
Publication status	Published - 1 Dec 2020
Externally published	Yes
MoE publication type	A1 Journal article-refereed

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10.1038/s41598-020-59091-3

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title = "Assessment of recombinant protein production in E. coli with Time-Gated Surface Enhanced Raman Spectroscopy (TG-SERS)",

abstract = "Time-Gated Surface-Enhanced Raman spectroscopy (TG-SERS) was utilized to assess recombinant protein production in Escherichia coli. TG-SERS suppressed the fluorescence signal from the biomolecules in the bacteria and the culture media. Characteristic protein signatures at different time points of the cell cultivation were observed and compared to conventional continuous wave (CW)-Raman with SERS. TG-SERS can distinguish discrete features of proteins such as the secondary structures and is therefore indicative of folding or unfolding of the protein. A novel method utilizing nanofibrillar cellulose as a stabilizing agent for nanoparticles and bacterial cells was used for the first time in order to boost the Raman signal, while simultaneously suppressing background signals. We evaluated the expression of hCNTF, hHspA1, and hHsp27 in complex media using the batch fermentation mode. HCNTF was also cultivated using EnBase in a fed-batch like mode. HspA1 expressed poorly due to aggregation problems within the cell, while hCNTF expressed in batch mode was correctly folded and protein instabilities were identified in the EnBase cultivation. Time-gated Raman spectroscopy showed to be a powerful tool to evaluate protein production and correct folding within living E. coli cells during the cultivation.",

author = "Martin K{\"o}gler and Jaakko Itkonen and Tapani Viitala and Casteleijn, {Marco G.}",

note = "Funding Information: We thank Dr. Wilbert Boelens of Raboud University (Nijmegen, the Netherlands) for providing us with the pET3a-Hsp27 plasmid containing the human Hsp27 gene. We also would like to thank Dr. Afsahi Ghazaleh (Aalto University, Finland) and Leena Pietil{\"a} (University of Helsinki, Finland) for their kind laboratory assistance and Regina Casteleijn-Osorno of AaltoEE (Finland) for comments that greatly improved the manuscript. Furthermore, we thank the Academy of Finland with FOULSENS-project (292253), the “Disrupting biotechnology to achieve high quality synthesis for biological drug development” Key-project (303884), and the VTT technical research centre of Finland for financial support. This work was funded and supported by Academy of Finland project 303884, 292253 and the VTT Technical Research Institute Finland. Publisher Copyright: {\textcopyright} 2020, The Author(s).",

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AU - Casteleijn, Marco G.

N1 - Funding Information: We thank Dr. Wilbert Boelens of Raboud University (Nijmegen, the Netherlands) for providing us with the pET3a-Hsp27 plasmid containing the human Hsp27 gene. We also would like to thank Dr. Afsahi Ghazaleh (Aalto University, Finland) and Leena Pietilä (University of Helsinki, Finland) for their kind laboratory assistance and Regina Casteleijn-Osorno of AaltoEE (Finland) for comments that greatly improved the manuscript. Furthermore, we thank the Academy of Finland with FOULSENS-project (292253), the “Disrupting biotechnology to achieve high quality synthesis for biological drug development” Key-project (303884), and the VTT technical research centre of Finland for financial support. This work was funded and supported by Academy of Finland project 303884, 292253 and the VTT Technical Research Institute Finland. Publisher Copyright: © 2020, The Author(s).

PY - 2020/12/1

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N2 - Time-Gated Surface-Enhanced Raman spectroscopy (TG-SERS) was utilized to assess recombinant protein production in Escherichia coli. TG-SERS suppressed the fluorescence signal from the biomolecules in the bacteria and the culture media. Characteristic protein signatures at different time points of the cell cultivation were observed and compared to conventional continuous wave (CW)-Raman with SERS. TG-SERS can distinguish discrete features of proteins such as the secondary structures and is therefore indicative of folding or unfolding of the protein. A novel method utilizing nanofibrillar cellulose as a stabilizing agent for nanoparticles and bacterial cells was used for the first time in order to boost the Raman signal, while simultaneously suppressing background signals. We evaluated the expression of hCNTF, hHspA1, and hHsp27 in complex media using the batch fermentation mode. HCNTF was also cultivated using EnBase in a fed-batch like mode. HspA1 expressed poorly due to aggregation problems within the cell, while hCNTF expressed in batch mode was correctly folded and protein instabilities were identified in the EnBase cultivation. Time-gated Raman spectroscopy showed to be a powerful tool to evaluate protein production and correct folding within living E. coli cells during the cultivation.

AB - Time-Gated Surface-Enhanced Raman spectroscopy (TG-SERS) was utilized to assess recombinant protein production in Escherichia coli. TG-SERS suppressed the fluorescence signal from the biomolecules in the bacteria and the culture media. Characteristic protein signatures at different time points of the cell cultivation were observed and compared to conventional continuous wave (CW)-Raman with SERS. TG-SERS can distinguish discrete features of proteins such as the secondary structures and is therefore indicative of folding or unfolding of the protein. A novel method utilizing nanofibrillar cellulose as a stabilizing agent for nanoparticles and bacterial cells was used for the first time in order to boost the Raman signal, while simultaneously suppressing background signals. We evaluated the expression of hCNTF, hHspA1, and hHsp27 in complex media using the batch fermentation mode. HCNTF was also cultivated using EnBase in a fed-batch like mode. HspA1 expressed poorly due to aggregation problems within the cell, while hCNTF expressed in batch mode was correctly folded and protein instabilities were identified in the EnBase cultivation. Time-gated Raman spectroscopy showed to be a powerful tool to evaluate protein production and correct folding within living E. coli cells during the cultivation.

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ER -

Assessment of recombinant protein production in E. coli with Time-Gated Surface Enhanced Raman Spectroscopy (TG-SERS)

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