Metabolic Fingerprint Analysis of Cytochrome b5-producing E. coli N4830-1 Using FT-IR Spectroscopy
Optimization of recombinant protein expression in bacteria is an important task in order to increase protein yield while maintaining the structural fidelity of the product. In this study, we employ Fourier transform infrared (FT-IR) spectroscopy as a high throughput metabolic fingerprinting approach...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-06-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.874247/full |
| _version_ | 1818235942675152896 |
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| author | Thanyaporn Tengsuttiwat Naheed Nazly Kaderbhai Joe Gallagher Royston Goodacre Howbeer Muhamadali |
| author_facet | Thanyaporn Tengsuttiwat Naheed Nazly Kaderbhai Joe Gallagher Royston Goodacre Howbeer Muhamadali |
| author_sort | Thanyaporn Tengsuttiwat |
| collection | DOAJ |
| description | Optimization of recombinant protein expression in bacteria is an important task in order to increase protein yield while maintaining the structural fidelity of the product. In this study, we employ Fourier transform infrared (FT-IR) spectroscopy as a high throughput metabolic fingerprinting approach to optimize and monitor cytochrome b5 (CYT b5) production in Escherichia coli N4830-1, as the heterologous host. Cyt b5 was introduced as a plasmid with between 0 and 6 copies under a strong promoter. The FT-IR spectroscopy results combined with multivariate chemometric analysis illustrated discriminations among culture conditions as well as revealing features that correlated to the different cytb5 gene copy numbers. The second derivative of the FT-IR spectral data allowed for the quantitative detection of Cyt b5 directly inside the intact cells without the need for extraction, and highlighted changes in protein secondary structure that was directly correlated to the cytb5 gene copy number and protein content, and was in complete agreement with quantitative findings of standard traditional techniques such as SDS–PAGE and western blot analysis. |
| first_indexed | 2024-12-12T12:01:59Z |
| format | Article |
| id | doaj.art-9db74e5df36845e6af589fa273c03dd8 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-12T12:01:59Z |
| publishDate | 2022-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-9db74e5df36845e6af589fa273c03dd82022-12-22T00:25:05ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-06-011310.3389/fmicb.2022.874247874247Metabolic Fingerprint Analysis of Cytochrome b5-producing E. coli N4830-1 Using FT-IR SpectroscopyThanyaporn Tengsuttiwat0Naheed Nazly Kaderbhai1Joe Gallagher2Royston Goodacre3Howbeer Muhamadali4Centre for Metabolomics Research, Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United KingdomInstitute of Biological, Environmental and Rural Sciences, Gogerddan Campus, Aberystwyth University, Aberystwyth, United KingdomInstitute of Biological, Environmental and Rural Sciences, Gogerddan Campus, Aberystwyth University, Aberystwyth, United KingdomCentre for Metabolomics Research, Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United KingdomCentre for Metabolomics Research, Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, United KingdomOptimization of recombinant protein expression in bacteria is an important task in order to increase protein yield while maintaining the structural fidelity of the product. In this study, we employ Fourier transform infrared (FT-IR) spectroscopy as a high throughput metabolic fingerprinting approach to optimize and monitor cytochrome b5 (CYT b5) production in Escherichia coli N4830-1, as the heterologous host. Cyt b5 was introduced as a plasmid with between 0 and 6 copies under a strong promoter. The FT-IR spectroscopy results combined with multivariate chemometric analysis illustrated discriminations among culture conditions as well as revealing features that correlated to the different cytb5 gene copy numbers. The second derivative of the FT-IR spectral data allowed for the quantitative detection of Cyt b5 directly inside the intact cells without the need for extraction, and highlighted changes in protein secondary structure that was directly correlated to the cytb5 gene copy number and protein content, and was in complete agreement with quantitative findings of standard traditional techniques such as SDS–PAGE and western blot analysis.https://www.frontiersin.org/articles/10.3389/fmicb.2022.874247/fullFT-IR spectroscopymetabolic fingerprintrecombinant protein productioncytochrome b5chemometrics |
| spellingShingle | Thanyaporn Tengsuttiwat Naheed Nazly Kaderbhai Joe Gallagher Royston Goodacre Howbeer Muhamadali Metabolic Fingerprint Analysis of Cytochrome b5-producing E. coli N4830-1 Using FT-IR Spectroscopy Frontiers in Microbiology FT-IR spectroscopy metabolic fingerprint recombinant protein production cytochrome b5 chemometrics |
| title | Metabolic Fingerprint Analysis of Cytochrome b5-producing E. coli N4830-1 Using FT-IR Spectroscopy |
| title_full | Metabolic Fingerprint Analysis of Cytochrome b5-producing E. coli N4830-1 Using FT-IR Spectroscopy |
| title_fullStr | Metabolic Fingerprint Analysis of Cytochrome b5-producing E. coli N4830-1 Using FT-IR Spectroscopy |
| title_full_unstemmed | Metabolic Fingerprint Analysis of Cytochrome b5-producing E. coli N4830-1 Using FT-IR Spectroscopy |
| title_short | Metabolic Fingerprint Analysis of Cytochrome b5-producing E. coli N4830-1 Using FT-IR Spectroscopy |
| title_sort | metabolic fingerprint analysis of cytochrome b5 producing e coli n4830 1 using ft ir spectroscopy |
| topic | FT-IR spectroscopy metabolic fingerprint recombinant protein production cytochrome b5 chemometrics |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.874247/full |
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