Relationship Between Recombinant Protein Expression and Host Metabolome As Determined by Two-dimensional NMR Spectroscopy

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2017 May 10

PMID 28486539

Citations 5

Authors

Young Kee Chae

Seol Hyun Kim

John L Markley

Affiliations

Soon will be listed here.

Abstract

Escherichia coli has been the most widely used host to produce large amounts of heterologous proteins. However, given an input plasmid DNA, E. coli may produce soluble protein, produce only inclusion bodies, or yield little or no protein at all. Many efforts have been made to surmount these problems, but most of them have involved time-consuming and labor-intensive trial-and-error. We hypothesized that different metabolomic fingerprints might be associated with different protein production outcomes. If so, then it might be possible to change the expression pattern by manipulating the metabolite environment. As a first step in testing this hypothesis, we probed a subset of the intracellular metabolites by partially labeling it with 13C-glucose. We tested 71 genes and identified 17 metabolites by employing the two-dimensional NMR spectroscopy. The statistical analysis showed that there existed the metabolite compositions favoring protein production. We hope that this work would help devise a systematic and predictive approach to the recombinant protein production.

Citing Articles

Designing a Secretory form of RTX-A as an Anticancer Toxin: An Approach.

Taheri-Anganeh M, Nezafat N, Gharibi S, Khatami S, Vahedi F, Shabaninejad Z Recent Pat Biotechnol. 2024; 18(4):332-343.

PMID: 38817010 DOI: 10.2174/0118722083267796231210060150.

In Search of Complementary Extraction Methods for Comprehensive Coverage of the Metabolome.

Gould H, Cheung W, Finnigan J, Munoz-Munoz J, Charnock S, Black G Metabolites. 2023; 13(9).

PMID: 37755290 PMC: 10535102. DOI: 10.3390/metabo13091010.

Identification of interaction partners using protein aggregation and NMR spectroscopy.

Chae Y, Shin H, Woo T PLoS One. 2022; 17(9):e0270058.

PMID: 36084098 PMC: 9462707. DOI: 10.1371/journal.pone.0270058.

Designing next generation recombinant protein expression platforms by modulating the cellular stress response in Escherichia coli.

Guleria R, Jain P, Verma M, Mukherjee K Microb Cell Fact. 2020; 19(1):227.

PMID: 33308214 PMC: 7730785. DOI: 10.1186/s12934-020-01488-w.

Identification of bioactive metabolites using activity metabolomics.

Rinschen M, Ivanisevic J, Giera M, Siuzdak G Nat Rev Mol Cell Biol. 2019; 20(6):353-367.

PMID: 30814649 PMC: 6613555. DOI: 10.1038/s41580-019-0108-4.

References

Xia J, Sinelnikov I, Han B, Wishart D . MetaboAnalyst 3.0--making metabolomics more meaningful. Nucleic Acids Res. 2015; 43(W1):W251-7. PMC: 4489235. DOI: 10.1093/nar/gkv380. View

Zhang Y, Howitt J, McCorkle S, Lawrence P, Springer K, Freimuth P . Protein aggregation during overexpression limited by peptide extensions with large net negative charge. Protein Expr Purif. 2004; 36(2):207-16. DOI: 10.1016/j.pep.2004.04.020. View

Bessette P, Aslund F, Beckwith J, Georgiou G . Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm. Proc Natl Acad Sci U S A. 1999; 96(24):13703-8. PMC: 24128. DOI: 10.1073/pnas.96.24.13703. View

Brown B, Hadley M, Page R . Heterologous high-level E. coli expression, purification and biophysical characterization of the spine-associated RapGAP (SPAR) PDZ domain. Protein Expr Purif. 2008; 62(1):9-14. DOI: 10.1016/j.pep.2008.07.003. View

Prinz W, Aslund F, Holmgren A, Beckwith J . The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J Biol Chem. 1997; 272(25):15661-7. DOI: 10.1074/jbc.272.25.15661. View

Sajed T, Marcu A, Ramirez M, Pon A, Guo A, Knox C . ECMDB 2.0: A richer resource for understanding the biochemistry of E. coli. Nucleic Acids Res. 2015; 44(D1):D495-501. PMC: 4702790. DOI: 10.1093/nar/gkv1060. View

Delaglio F, Grzesiek S, Vuister G, Zhu G, Pfeifer J, Bax A . NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995; 6(3):277-93. DOI: 10.1007/BF00197809. View

Akiyama K, Chikayama E, Yuasa H, Shimada Y, Tohge T, Shinozaki K . PRIMe: a Web site that assembles tools for metabolomics and transcriptomics. In Silico Biol. 2008; 8(3-4):339-45. View

Chen Y, Song J, Sui S, Wang D . DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli. Protein Expr Purif. 2004; 32(2):221-31. DOI: 10.1016/S1046-5928(03)00233-X. View

10.

Spraul M, Schutz B, Humpfer E, Mortter M, Schafer H, Koswig S . Mixture analysis by NMR as applied to fruit juice quality control. Magn Reson Chem. 2009; 47 Suppl 1:S130-7. DOI: 10.1002/mrc.2528. View

11.

Blommel P, Martin P, Seder K, Wrobel R, Fox B . Flexi vector cloning. Methods Mol Biol. 2008; 498:55-73. PMC: 5957505. DOI: 10.1007/978-1-59745-196-3_4. View

12.

De Marco V, Stier G, Blandin S, de Marco A . The solubility and stability of recombinant proteins are increased by their fusion to NusA. Biochem Biophys Res Commun. 2004; 322(3):766-71. DOI: 10.1016/j.bbrc.2004.07.189. View

13.

Malakhov M, Mattern M, Malakhova O, Drinker M, Weeks S, Butt T . SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. J Struct Funct Genomics. 2004; 5(1-2):75-86. DOI: 10.1023/B:JSFG.0000029237.70316.52. View

14.

Wang Y, Ayrapetov M, Lin X, Sun G . A new strategy to produce active human Src from bacteria for biochemical study of its regulation. Biochem Biophys Res Commun. 2006; 346(2):606-11. DOI: 10.1016/j.bbrc.2006.05.180. View

15.

Studier F . Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005; 41(1):207-34. DOI: 10.1016/j.pep.2005.01.016. View

16.

Chae Y, Kim S, Ellinger J, Markley J . Tracing Metabolite Footsteps of Along the Time Course of Recombinant Protein Expression by Two-Dimensional NMR Spectroscopy. Bull Korean Chem Soc. 2013; 33(12):4041-4046. PMC: 3686544. DOI: 10.5012/bkcs.2012.33.12.4041. View

17.

Chae Y, Kim S, Ellinger J, Markley J . Dosage Effects of Salt and pH Stresses on as Monitored Metabolites by Using Two Dimensional NMR Spectroscopy. Bull Korean Chem Soc. 2015; 34(12):3602-3608. PMC: 4311725. DOI: 10.5012/bkcs.2013.34.12.3602. View

18.

Lewis I, Schommer S, Hodis B, Robb K, Tonelli M, Westler W . Method for determining molar concentrations of metabolites in complex solutions from two-dimensional 1H-13C NMR spectra. Anal Chem. 2007; 79(24):9385-90. PMC: 2533272. DOI: 10.1021/ac071583z. View

19.

Chae Y, Kim S, Seong Hyun J . Probing Metabolite Space of Escherichia coli via Growth Medium Composition as Monitored by Two-Dimensional NMR Spectroscopy. Chem Biodivers. 2015; 12(6):925-36. DOI: 10.1002/cbdv.201400200. View

20.

Ritz D, Beckwith J . Roles of thiol-redox pathways in bacteria. Annu Rev Microbiol. 2001; 55:21-48. DOI: 10.1146/annurev.micro.55.1.21. View