CoChat
Sign Up
Cited by 260Protein purification and stabilityRedox biology and oxidative stressBacterial Genetics and Biotechnology

The DOI matches its Crossref record; 31 of the 31 references it cites were found in a registry or an index.

Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli

Open access
Nguyen, Van Dat, Hatahet, Feras, Salo, Kirsi EH, Enlund, Eveliina, Zhang, Chi, Ruddock, Lloyd W·2011·Microbial Cell Factories
DOI 10.1186/1475-2859-10-1PMC PMC3022669
Open this paper in CoChat — free accountOpen PDF

Abstract

Background: Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes. Results: Here we show that the introduction of Erv1p, a sulfhydryl oxidase and a disulfide isomerase allows the efficient formation of natively folded eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli. The production of disulfide bonded proteins was also aided by the use of an appropriate fusion protein to keep the folding intermediates soluble and by choice of media. By combining the pre-expression of a sulfhydryl oxidase and a disulfide isomerase with these other factors, high level expression of even complex disulfide bonded eukaryotic proteins is possible Conclusions: Our results show that the production of eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli is possible. The required exogenous components can be put onto a single plasmid vector allowing facile transfer between different prokaryotic strains. These results open up new avenues for the use of E. coli as a microbial cell factory.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1186/1475-2859-10-1

Cites

31 of 31 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1 Production of vtPA in the cytoplasm of E. coli. A) Relative yields of active vtPA produced in LB media at 30°C, normalized to the equivalent of the best previously reported system (a Δgor ΔtrxB strain with co-expression of mature DsbC) and shown as mean ± s.d. (n = 4-12; see additional file 1: Table of vtPA production and activity data). Strains: BL = BL21 (DE3) pLysSRARE; RG = rosetta-gami; OG = origami. When present Erv1p and mature DsbC are co-expressed from a polycistronic vector. B) SDS-PAGE analysis of MBP-fusion vtPA in LB media. T = total E. coli lysate, S = soluble fraction. The position of MBP-vtPA is marked with an arrow. Note the difference in expression levels in rosetta-gami compared with origami due to the expression of rare tRNAs.
Figure 1 Production of vtPA in the cytoplasm of E. coli. A) Relative yields of active vtPA produced in LB media at 30°C, normalized to the equivalent of the best previously reported system (a Δgor ΔtrxB strain with co-expression of mature DsbC) and shown as mean ± s.d. (n = 4-12; see additional file 1: Table of vtPA production and activity data). Strains: BL = BL21 (DE3) pLysSRARE; RG = rosetta-gami; OG = origami. When present Erv1p and mature DsbC are co-expressed from a polycistronic vector. B) SDS-PAGE analysis of MBP-fusion vtPA in LB media. T = total E. coli lysate, S = soluble fraction. The position of MBP-vtPA is marked with an arrow. Note the difference in expression levels in rosetta-gami compared with origami due to the expression of rare tRNAs.CC BY · PubMed Central
Figure 2 Design of the plasmids for pre- and co-expression of Erv1p and/or a protein disulfide isomerase. Co-expression is based on a polycistronic vector based on pET23 with a T7 promoter/terminator and IPTG induction. Pre-expression is based on cloning the Erv1p +/- the disulfide isomerase into an arabinose inducible system cloned into pLysS. Since the vector map for pLysSRARE is not available we were unable to clone into this vector and hence when using a Δgor ΔtrxB strain the co-expression studies are done in an origami rather than rosetta-gami background.
Figure 2 Design of the plasmids for pre- and co-expression of Erv1p and/or a protein disulfide isomerase. Co-expression is based on a polycistronic vector based on pET23 with a T7 promoter/terminator and IPTG induction. Pre-expression is based on cloning the Erv1p +/- the disulfide isomerase into an arabinose inducible system cloned into pLysS. Since the vector map for pLysSRARE is not available we were unable to clone into this vector and hence when using a Δgor ΔtrxB strain the co-expression studies are done in an origami rather than rosetta-gami background.CC BY · PubMed Central
Figure 3 The effect of co- vs pre-expression of folding factors on the production of vtPA in the cytoplasm of E. coli. Relative yields of active MBP-vtPA produced in LB media at 30°C, normalized to the equivalent of the best previously reported system (a Δgor ΔtrxB strain with co-expression of mature DsbC) and shown as mean ± s.d. (n = 4-12; see additional file 1: Table of vtPA production and activity data). Strains: RG = rosetta-gami; OG = origami. For Erv1p and mature DsbC expression + = co-expression while +p = pre-expression. Note the increase in vtPA activity with pre-expression comes despite the significant reduction in vtPA expression levels in rosetta-gami compared with origami due to the expression of rare tRNAs (figure 1B).
Figure 3 The effect of co- vs pre-expression of folding factors on the production of vtPA in the cytoplasm of E. coli. Relative yields of active MBP-vtPA produced in LB media at 30°C, normalized to the equivalent of the best previously reported system (a Δgor ΔtrxB strain with co-expression of mature DsbC) and shown as mean ± s.d. (n = 4-12; see additional file 1: Table of vtPA production and activity data). Strains: RG = rosetta-gami; OG = origami. For Erv1p and mature DsbC expression + = co-expression while +p = pre-expression. Note the increase in vtPA activity with pre-expression comes despite the significant reduction in vtPA expression levels in rosetta-gami compared with origami due to the expression of rare tRNAs (figure 1B).CC BY · PubMed Central
Figure 4 Effects of media on vtPA production in the cytoplasm of E. coli. Panel A) Relative fold activity of vtPA produced in EnBase media at 30°C, normalized to the equivalent of the best previously reported system (rosetta-gami with co-expression of mature DsbC) and shown as mean ± s.d. (n = 2-6; see additional file 1: Table of vtPA production and activity data). Strains: BL = BL21 (DE3) pLysSRARE; RG = rosetta-gami; OG = origami. For Erv1p and mature DsbC expression + = co-expression while +p = pre-expression. Panel B) Relative yields of active vtPA made as an MBP fusion protein grown in LB and EnBase media in shake flasks at 30°C. Nomenclature as per the previous panel.
Figure 4 Effects of media on vtPA production in the cytoplasm of E. coli. Panel A) Relative fold activity of vtPA produced in EnBase media at 30°C, normalized to the equivalent of the best previously reported system (rosetta-gami with co-expression of mature DsbC) and shown as mean ± s.d. (n = 2-6; see additional file 1: Table of vtPA production and activity data). Strains: BL = BL21 (DE3) pLysSRARE; RG = rosetta-gami; OG = origami. For Erv1p and mature DsbC expression + = co-expression while +p = pre-expression. Panel B) Relative yields of active vtPA made as an MBP fusion protein grown in LB and EnBase media in shake flasks at 30°C. Nomenclature as per the previous panel.CC BY · PubMed Central
Figure 5 Production of BPTI in the cytoplasm of E. coli. Analysis of mature BPTI wild-type and mutants which mimic the two disulfide folding intermediates produced in the cytoplasm of origami with pre-expression of Erv1p and PDI in LB media at 30°C. Panel A) Coomassie stained SDS-PAGE. T = total E. coli lysate; S = soluble fraction. The position of BPTI is marked with an arrow. Panel B) rpHPLC analysis of purified BPTI mutants under conditions which allow identification of the different folding intermediates. Upper trace (displaced 50 mAU) C30A/C51A mutant, lower trace C14A/C38A mutant. A single peak with the elution point being equivalent to the relevant two disulfide containing wild-type folding intermediate was obtained for both proteins.
Figure 5 Production of BPTI in the cytoplasm of E. coli. Analysis of mature BPTI wild-type and mutants which mimic the two disulfide folding intermediates produced in the cytoplasm of origami with pre-expression of Erv1p and PDI in LB media at 30°C. Panel A) Coomassie stained SDS-PAGE. T = total E. coli lysate; S = soluble fraction. The position of BPTI is marked with an arrow. Panel B) rpHPLC analysis of purified BPTI mutants under conditions which allow identification of the different folding intermediates. Upper trace (displaced 50 mAU) C30A/C51A mutant, lower trace C14A/C38A mutant. A single peak with the elution point being equivalent to the relevant two disulfide containing wild-type folding intermediate was obtained for both proteins.CC BY · PubMed Central
Figure 6 Analysis of mature human resistin expressed in the cytoplasm of E. coli at 30°C in LB. Panel A) Coomassie stained SDS-PAGE analysis of purified MBPx-resistin indicates production of the disulfide linked dimer increases with pre-expression of Erv1p and DsbC in both wild-type and Δgor ΔtrxB strains. The molecular weights (in kDa) of the markers (mw) are indicated as are the positions of the monomeric and dimeric forms of MBPx-resistin. Panel B) Coomassie stained SDS-PAGE analysis of mature resistin produced in origami with pre-expression of Erv1p and DsbC. S = soluble fraction of E. coli lysates; P = Purified MBPx-resistin from an amylose column; D = Purified material that has been digested with factor Xa to release MBP and resistin (marked with *). Note that under non-reducing conditions the resistin is a disulfide linked homo-dimer. The molecular weights (in kDa) of the markers (mw) are indicated as is the position of MBP post digestion. The position of the MBP-resistin fusion is indicated with an arrow.
Figure 6 Analysis of mature human resistin expressed in the cytoplasm of E. coli at 30°C in LB. Panel A) Coomassie stained SDS-PAGE analysis of purified MBPx-resistin indicates production of the disulfide linked dimer increases with pre-expression of Erv1p and DsbC in both wild-type and Δgor ΔtrxB strains. The molecular weights (in kDa) of the markers (mw) are indicated as are the positions of the monomeric and dimeric forms of MBPx-resistin. Panel B) Coomassie stained SDS-PAGE analysis of mature resistin produced in origami with pre-expression of Erv1p and DsbC. S = soluble fraction of E. coli lysates; P = Purified MBPx-resistin from an amylose column; D = Purified material that has been digested with factor Xa to release MBP and resistin (marked with *). Note that under non-reducing conditions the resistin is a disulfide linked homo-dimer. The molecular weights (in kDa) of the markers (mw) are indicated as is the position of MBP post digestion. The position of the MBP-resistin fusion is indicated with an arrow.CC BY · PubMed Central
Figure 7 Screening for the production of for other human proteins using Erv1p plus a disulfide isomerase pre-expression system. Panel A) Coomassie stained reducing SDS-PAGE analysis of mature human proteins produced in the cytoplasm of BL21 (DE3) pLysS with different growth media. T = total E. coli lysate S = soluble fraction. The positions of the proteins are marked with arrows. The data shown is from Ero1α with pre-expression of Erv1p and mature PDI; MBP-tagged CSF3 with pre-expression of Erv1p and mature DsbC and co-expression of Erv1p and mature PDI; MBPx-tagged BMP4 with pre-and co-expression of Erv1p and DsbC; MBPx-light chain of enterokinase Cys896Ser with pre-expression of Erv1p and DsbC. Many other variants and combinations of pre- and co-expression conditions have been tested (see additional file 2: Table of vectors used in this study). For all four of these proteins under all conditions tested there is a concomitant increase in the number of disulfide bonds and yields of monomeric protein upon co- or pre-expression of Erv1p and a disulfide isomerase. Panel B) Reducing SDS-PAGE analysis of mature human proteins produced in the cytoplasm of origami in EnBase media with pre-expression of Erv1p and DsbC. T = total E. coli lysate, S = soluble fraction, P = protein purified using an NTA-spin column or amylose column. The molecular weights (in kDa) of the markers (mw) are indicated.
Figure 7 Screening for the production of for other human proteins using Erv1p plus a disulfide isomerase pre-expression system. Panel A) Coomassie stained reducing SDS-PAGE analysis of mature human proteins produced in the cytoplasm of BL21 (DE3) pLysS with different growth media. T = total E. coli lysate S = soluble fraction. The positions of the proteins are marked with arrows. The data shown is from Ero1α with pre-expression of Erv1p and mature PDI; MBP-tagged CSF3 with pre-expression of Erv1p and mature DsbC and co-expression of Erv1p and mature PDI; MBPx-tagged BMP4 with pre-and co-expression of Erv1p and DsbC; MBPx-light chain of enterokinase Cys896Ser with pre-expression of Erv1p and DsbC. Many other variants and combinations of pre- and co-expression conditions have been tested (see additional file 2: Table of vectors used in this study). For all four of these proteins under all conditions tested there is a concomitant increase in the number of disulfide bonds and yields of monomeric protein upon co- or pre-expression of Erv1p and a disulfide isomerase. Panel B) Reducing SDS-PAGE analysis of mature human proteins produced in the cytoplasm of origami in EnBase media with pre-expression of Erv1p and DsbC. T = total E. coli lysate, S = soluble fraction, P = protein purified using an NTA-spin column or amylose column. The molecular weights (in kDa) of the markers (mw) are indicated.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 31 references

Cited by

Sorted by citations

Items 201–259 of 259

Related

See all

Metadata

Authors
Nguyen, V. D., Hatahet, F., Salo, K. E., Enlund, E., Zhang, C., & Ruddock, L. W.
Affiliations
University of Oulu (FI)
Corresponding author
Ruddock, Lloyd W
ORCID
Venue
Microbial Cell Factories
Publisher
Springer Science and Business Media LLC
Published
2011-01-07
Volume
10
Issue
1
Pages
1-1
ISSN
1475-2859 (ISSN-L)
DOI
10.1186/1475-2859-10-1
PMC
PMC3022669
OpenAlex
W2135101596
Cited by
260
References
31
Citations by year
2026: 7 · 2025: 8 · 2024: 12 · 2023: 20 · 2022: 14 · 2021: 18 · 2020: 19 · 2019: 13 · 2018: 16 · 2017: 20 · 2016: 18 · 2015: 18 · 2014: 24 · 2013: 25 · 2012: 17
FWCI
10.01
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Microbial Cell Factories (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Academy of Finland
  • Tekes
  • Oulun Yliopisto
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
sulfhydryl oxidase, disulfide bond formation, protein disulfide isomerase, fusion protein, microbial cell factories, recombinant protein expression
MeSH terms
Cytoplasm, Disulfides, Escherichia coli, Genetic Engineering, Humans, Oxidoreductases, Protein Processing, Post-Translational, Recombinant Proteins, Gene Expression, Protein Disulfide-Isomerases, Protein Transport, Oxidoreductases Acting on Sulfur Group Donors

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review