Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Suppressing glucose uptake and acetic acid production increases membrane protein overexpression in Escherichia coli.
KTH, School of Biotechnology (BIO), Bioprocess Technology.
KTH, School of Biotechnology (BIO), Bioprocess Technology.ORCID iD: 0000-0002-6979-0069
2011 (English)In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 10, no 1, 35- p.Article in journal (Refereed) Published
Abstract [en]

The production of integral membrane spanning proteins (IMP's) constitutes a bottleneck in pharmaceutical development. It was long considered that the state-of-the-art was to produce the proteins as inclusion bodies using a powerful induction system. However, the quality of the protein was compromised and the production of a soluble protein that is incorporated into the membrane from which it is extracted is now considered to be a better method. Earlier research has indicated that a slower rate of protein synthesis might overcome the tendency to form inclusion bodies. We here suggest the use of a set of E. coli mutants characterized by a slower rate of growth and protein synthesis as a tool for increasing the amount of soluble protein in high- throughput protein production processes. RESULTS: A set of five IMP's was chosen which were expressed in three mutants and the corresponding WT cell (control). The mutations led to three different substrate uptake rates, two of which were considerably slower than that of the wild type. Using the mutants, we were able to express three out of the five membrane proteins. Most successful was the mutant growing at 50% of the wild type growth rate. A further effect of a low growth rate is a low acetic acid formation, and we believe that this is a possible reason for the better production. This hypothesis was further supported by expression from the BL21(DE3) strain, using the same plasmid. This strain grows at a high growth rate but nevertheless yields only small amounts of acetic acid. This strain was also able to express three out of the five IMP's, although at lower quantities. CONCLUSIONS: The use of mutants that reduce the specific substrate uptake rate seems to be a versatile tool for overcoming some of the difficulties in the production of integral membrane spanning proteins. A set of strains with mutations in the glucose uptake system and with a lower acetic acid formation were able to produce three out of five membrane proteins that it was not possible to produce with the corresponding wild type.

Place, publisher, year, edition, pages
2011. Vol. 10, no 1, 35- p.
Keyword [en]
AIDA-autotransporter, Escherichia coli, fed-batch, glucose uptake rate, integral membrane proteins, outer membrane proteins, periplasmic retention, phosphotransferase system, recombinant proteins, specific growth rate, surface expression
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-34433DOI: 10.1186/1475-2859-10-35ISI: 000291990500001PubMedID: 21586123Scopus ID: 2-s2.0-79955953366OAI: oai:DiVA.org:kth-34433DiVA: diva2:421359
Note
Updated from submitted to published. QC 20110711Available from: 2011-06-08 Created: 2011-06-08 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Impact of glucose uptake rate on recombinant protein production in Escherichia coli
Open this publication in new window or tab >>Impact of glucose uptake rate on recombinant protein production in Escherichia coli
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli.

E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures.  

The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation.

For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm.

The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:18
Keyword
AIDA-autotransporter, Escherichia coli, fed-batch, glucose uptake rate, integral membrane proteins, outer membrane proteins, periplasmic retention, phosphotransferase system, recombinant proteins, specific growth rate, surface expression
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-34019 (URN)978-91-7415-994-3 (ISBN)
Public defence
2011-06-15, FB52, AlbaNova, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20110608Available from: 2011-06-08 Created: 2011-05-23 Last updated: 2012-02-14Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMedScopus

Authority records BETA

Larsson, Gen

Search in DiVA

By author/editor
Backlund, EmmaLarsson, Gen
By organisation
Bioprocess Technology
In the same journal
Microbial Cell Factories
Biological Sciences

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 70 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf