Abstract
Multi-parameter flow cytometry was used to monitor the formation of promegapoietin (PMP) inclusion bodies during a high cell density Escherichia coli fed-batch fermentation process. Inclusion bodies were labelled with a primary antibody and then with a secondary fluorescent antibody. Using this method it was possible to detect PMP inclusion body formation with a high specificity and it was possible to monitor the increased accumulation of the protein with process time (6–48 mg PMP/g CDW) whilst highlighting population heterogeneity.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
B Allman R Hann A Manchess D Lloyd (1992) ArticleTitleCharacterisation of bacteria by multiparameter flow cytometry J. Appl. Bacteriol 73 438–444 Occurrence Handle1447060
CD Boswell AW Nienow NK Gill S Kocharunchitt CJ Hewitt (2003) ArticleTitleThe impact of fluid mechanical stress on Saccharomyces cerevisiae cells during continuous cultivation in an agitated, aerated bioreactor; its implications for mixing in the brewing process and aerobic fermentations Trans. I. Chem E., Part C 81 23–32
P Fouchet C Manin H Richard G Frelat JN Barbotin (1994) ArticleTitleFlow cytometry studies of recombinant Escherichia coli in batch and continuous cultures: DNA and RNA contents; light-scatter parameters Appl. Microbiol. Biot 41 584–590
CJ Hewitt G Nebe-von-Caron (2001) ArticleTitleAn industrial application of multiparameter flow cytometry: Assessment of cell physiological state and its application to the study of microbial fermentations Cytometry 44 179–187 Occurrence Handle10.1002/1097-0320(20010701)44:3<179::AID-CYTO1110>3.0.CO;2-D Occurrence Handle11429768
CJ Hewitt G Nebe-von-Caron (2004) ArticleTitleThe application of multi-parameter flow cytometry to monitor individual microbial cell physiological state Adv. Biochem. Eng./Biotechnol 89 197–223
F Lavergne-Mazeau A Maftah Y Centatiempo R Julien (1996) ArticleTitleLinear correlation between bacterial overexpression of recombinant peptides and light scatter Appl. Environ. Microb 62 3042–3046
G Lewis IW Taylor AW Nienow CJ Hewitt (2004) ArticleTitleThe application of multi-parameter flow cytometry to the study of recombinant Escherichia coli batch fermentation processes J. Ind. Microbiol. Biotechol 31 311–322
W Martinet D Plas ParticleVan den H Raes R Reekmans R Contreras (1999) ArticleTitleBax-induced cell death in Pichia pastoris Biotechnol. Lett 21 821–829 Occurrence Handle10.1023/A:1005586614543
A Mukhopadhyay (1997) ArticleTitleInclusion bodies and purification of proteins in biologically active forms Adv. Biochem. Eng/Biotechnol 56 61–109
G Nebe-von Caron RA Badley (1995) ArticleTitleViability assessment of bacteria in mixed populations using flow cytometry J. Microsc 179 55–66
G Nebe-von Caron R Badley (1996) Bacterial characterisation by flow cytometry M Al-Rubeai AN Emery (Eds) Flow Cytometry Applications in Cell Culture Marcel Dekker New York 257–290
G Nebe-von-Caron PJ Stephens CJ Hewitt JR Powell RA Badley (2000) ArticleTitleAnalysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting J. Microbiol. Meth 42 97–114 Occurrence Handle10.1016/S0167-7012(00)00181-0
MG Obukowicz NR Staten GG Krivi (1992) ArticleTitleEnhanced heterologous gene expression in novel rpoH mutants of Escherichia coli Appl. Environ. Microb 58 1511–1523
A Reis T Lopes da Silva CA Kent M Kosseva JC Roseiro CJ Hewitt (2005) ArticleTitleMonitoring population dynamics of the thermophilic Bacillus licheniformis CCMI 1034 in batch and continuous cultures using multi-parameter flow cytometry J. Biotechnol 115 199–210 Occurrence Handle10.1016/j.jbiotec.2004.08.005 Occurrence Handle15607238
BR Robertson DK Button AL Koch (1998) ArticleTitleDetermination of the biomasses of small bacteria at low concentrations in a mixture of species with forward light scatter measurements by flow cytometry Appl Environ. Microb 64 3900–3909
HM Shapiro (2000) ArticleTitleMicrobial analysis at the single-cell level: tasks and techniques J Microbiol Meth 42 3–16 Occurrence Handle10.1016/S0167-7012(00)00167-6
H Sundström F Wållberg E Ledung B Norrman CJ Hewitt SO Enfors (2004) ArticleTitleSegregation to non-dividing cells in recombinant Escherichia coli fed-batch fermentation processes Biotechnol. Lett 26 1533–1539 Occurrence Handle10.1023/B:BILE.0000044458.29147.75 Occurrence Handle15604793
J Vives-Rego P Lebaron G Nebe-von-Caron (2000) ArticleTitleCurrent and future applications of flow cytometry in aquatic microbiology FEMS Microbiol Rev 24 429–448 Occurrence Handle10.1016/S0168-6445(00)00033-4 Occurrence Handle10978545
KD Wittrup MB Mann DM Fenton LB Tsai JE Bailey (1988) ArticleTitleSingle-cell light scatter as a probe of refractile body formation in recombinant Escherichia coli Bio/Technology 6 423–426 Occurrence Handle10.1038/nbt0488-423
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Wållberg, F., Sundström, H., Ledung, E. et al. Monitoring and quantification of inclusion body formation in Escherichia coli by multi-parameter flow cytometry. Biotechnol Lett 27, 919–926 (2005). https://doi.org/10.1007/s10529-005-7184-6
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s10529-005-7184-6