The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis

Lenz P, Hilgers F, Burmeister A, Zimmermann L, Volkenborn K, Grünberger A, Kohlheyer D, Drepper T, Jaeger K-E, Knapp A (2021)
Microbial cell factories 20(1): 174.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
Download
Es wurden keine Dateien hochgeladen. Nur Publikationsnachweis!
Autor*in
Lenz, Patrick; Hilgers, Fabienne; Burmeister, Alina; Zimmermann, Leonie; Volkenborn, Kristina; Grünberger, AlexanderUniBi; Kohlheyer, Dietrich; Drepper, Thomas; Jaeger, Karl-Erich; Knapp, Andreas
Abstract / Bemerkung
BACKGROUND: Bacillussubtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh beta-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP.; RESULTS: As proof of concept, the GFP11-tag was fused C-terminally to the E. coli beta-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B.subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4-12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity.; CONCLUSION: Our results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B.subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level. © 2021. The Author(s).
Erscheinungsjahr
2021
Zeitschriftentitel
Microbial cell factories
Band
20
Ausgabe
1
Art.-Nr.
174
eISSN
1475-2859
Page URI
https://pub.uni-bielefeld.de/record/2957513

Zitieren

Lenz P, Hilgers F, Burmeister A, et al. The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis. Microbial cell factories. 2021;20(1): 174.
Lenz, P., Hilgers, F., Burmeister, A., Zimmermann, L., Volkenborn, K., Grünberger, A., Kohlheyer, D., et al. (2021). The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis. Microbial cell factories, 20(1), 174. https://doi.org/10.1186/s12934-021-01663-7
Lenz, P., Hilgers, F., Burmeister, A., Zimmermann, L., Volkenborn, K., Grünberger, A., Kohlheyer, D., Drepper, T., Jaeger, K. - E., and Knapp, A. (2021). The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis. Microbial cell factories 20:174.
Lenz, P., et al., 2021. The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis. Microbial cell factories, 20(1): 174.
P. Lenz, et al., “The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis”, Microbial cell factories, vol. 20, 2021, : 174.
Lenz, P., Hilgers, F., Burmeister, A., Zimmermann, L., Volkenborn, K., Grünberger, A., Kohlheyer, D., Drepper, T., Jaeger, K.-E., Knapp, A.: The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis. Microbial cell factories. 20, : 174 (2021).
Lenz, Patrick, Hilgers, Fabienne, Burmeister, Alina, Zimmermann, Leonie, Volkenborn, Kristina, Grünberger, Alexander, Kohlheyer, Dietrich, Drepper, Thomas, Jaeger, Karl-Erich, and Knapp, Andreas. “The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis”. Microbial cell factories 20.1 (2021): 174.

Export

Markieren/ Markierung löschen
Markierte Publikationen

Open Data PUB

Web of Science

Dieser Datensatz im Web of Science®

Quellen

PMID: 34488765
PubMed | Europe PMC

Suchen in

Google Scholar