Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • 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
Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Jan-Willem de Gier)
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Cornell University, Department of Plant Biology.
Show others and affiliations
2008 (English)In: Methods, ISSN 1095-9130, Vol. 46, no 2, 48-53 p.Article in journal (Refereed) Published
Abstract [en]

In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS–PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS–PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS–PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS–PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS–PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.

Place, publisher, year, edition, pages
2008. Vol. 46, no 2, 48-53 p.
Keyword [en]
rane protein, Protein complex, Two-dimensional blue native SDS–PAGE, Comparative proteomics, E. coli
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-17270DOI: 10.1016/j.ymeth.2008.06.017ISI: 000261367600002PubMedID: 18674622OAI: oai:DiVA.org:su-17270DiVA: diva2:183791
Available from: 2009-01-12 Created: 2009-01-12 Last updated: 2011-06-15Bibliographically approved
In thesis
1. Membrane Protein Biogenesis in Escherichia coli: A proteomics approach
Open this publication in new window or tab >>Membrane Protein Biogenesis in Escherichia coli: A proteomics approach
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the bacterium Escherichia coli all proteins are synthesized in the cytoplasm. However, at least 25% of them then need to be exported to another cellular compartment where they exert their function. E. coli has several different targeting pathways to ensure the correct localization of its proteins. When a nascent chain emerges at the ribosomal tunnel exit the signal recognition particle (SRP) can bind to it. The ribosomal nascent chain -SRP- complex is then targeted either to the Sec-translocon or the YidC insertase at the cytoplasmic membrane. Integral cytoplasmic membrane proteins are inserted into the lipid bilayer whilst periplasmic and outer membrane proteins are translocated across the membrane. In order to be fully functional, proteins need to be both correctly localized and folded. In many cases, they also assemble into complexes with other proteins or co-factors. In my thesis I will present an improved protocol for two-dimensional blue native/SDS-PAGE (2D BN/SDS-PAGE) that makes it very suitable to study the biogenesis of integral cytoplasmic membrane proteins and especially the complexes in the cytoplasmic membrane. Using this and other methods I have studied the biogenesis of integral cytoplasmic membrane proteins and other exported proteins. This has been done on a global scale in mutant strains where the SRP-targeting pathway, the Sec-translocon and the integral cytoplasmic membrane chaperone/insertase YidC have been compromised.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2010. 99 p.
Keyword
SRP, YidC, SecE, Membrane protein biogenesis, protein targeting, 2D BN/SDS-PAGE
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-36851 (URN)978-91-7447-008-6 (ISBN)
Public defence
2010-02-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2010-02-04 Created: 2010-01-27 Last updated: 2010-01-28Bibliographically approved
2. Into the Membrane and Beyond: Improving Membrane Protein Over-Expression in Escherichia coli
Open this publication in new window or tab >>Into the Membrane and Beyond: Improving Membrane Protein Over-Expression in Escherichia coli
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins fulfil a wide variety of essential functions in the cell and many are (potential) drug targets. Since their natural abundance is usually very low, most membrane proteins have to be over-expressed for functional and structural studies. T7 RNA polymerase (T7RNAP) based Escherichia coli strains, like BL21(DE3), are very popular protein production hosts. Unfortunately, over-expression of membrane proteins in E. coli is usually toxic to the cells. During my Ph.D. I have tried to understand the reasons for this toxicity by studying the consequences of membrane protein over-expression using a combination of proteomics and more focused biochemical and genetic methods. First, we had to improve the existing 2D BN/SDS-PAGE protocol to perform reliable comparative analysis of membrane proteomes. With the new protocol I have studied the effects of the expression of membrane proteins, including the human KDEL receptor, on BL21(DE3) and its derivatives, C41(DE3) and C43(DE3) (a.k.a. the Walker strains). The latter two were isolated for their improved membrane protein over-expression characteristics. Saturation of the Sec translocon, a cytoplasmic membrane associated protein conducting channel that mediates the insertion/biogenesis of membrane proteins, appeared to be the prime reason for the toxicity of membrane protein over-expression. Therefore, it was not surprising that we have identified mutations in the promoter governing the expression of the T7RNAP in the C41(DE3) and C43(DE3) strains that weaken it compared to the one in BL21(DE3). Based on this observation, we have engineered a plasmid (pLemo) with the gene encoding the natural inhibitor of T7RNAP, T7 lysozyme, under the control of the titratable rhamnose promoter. With the help of this plasmid the activity of the T7RNAP can be precisely set thereby avoiding saturation of the Sec translocon upon membrane protein over-expression. However, we have identified more changes in the Walker strains. Notable examples are the up regulation of peptide transporters in C41(DE3) and the expression of the Lon protease in C43(DE3). To study peptide import in E. coli I have characterized the in C41(DE3) strongly up regulated periplasmic binding protein OppA using a combination of biochemical and structural methods. The obtained data have resulted in many leads and ideas to further improve membrane protein over-expression yields in E. coli.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. 79 p.
Keyword
Escherichia coli, membrane protein over-expression, proteomics, T7 RNA polymerase, peptide transport, strain engineering
National Category
Biochemistry and Molecular Biology Physical Chemistry
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-57971 (URN)978-91-7447-295-0 (ISBN)
Public defence
2011-09-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.Available from: 2011-08-11 Created: 2011-05-25 Last updated: 2011-06-15Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Klepsch, MirjamSchlegel, SusanWickström, DavidPersson, Jan-Olovde Gier, Jan-Willem
By organisation
Department of Biochemistry and BiophysicsDepartment of Mathematics
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 87 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • 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