Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli.

ABSTRACT: Thionins are antimicrobial plant peptides produced as preproproteins consisting of a signal peptide, the thionin domain, and a so-called acidic domain. Only thionin itself has been isolated from plants. To study the processing of the precursor, it has to be produced in a heterologous system. Since both domains contain several cysteines and, due to the known antimicrobial activity of the thionin, we tested the expression of all four Arabidopsis proproteins as fusion proteins. Periplasmic expression as fusion with maltose binding protein was not successful but cytoplasmic expression as His-tagged TRX fusion proteins with a TEV recognition sequence resulted in proteins of correct size. Use of the SHuffle strain C3030 further improved the expression. Fusion proteins inhibited growth of Escherichia coli. They could be cleaved by TEV protease, releasing authentic proproteins without any additional amino acid at the N-terminus.

SUBMITTER: Abbas A

PROVIDER: S-EPMC3677976 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli.

Abbas Amjad A Plattner Stephan S Shah Kausar Hussain KH Bohlmann Holger H

Biotechnology letters 20130321 7

Thionins are antimicrobial plant peptides produced as preproproteins consisting of a signal peptide, the thionin domain, and a so-called acidic domain. Only thionin itself has been isolated from plants. To study the processing of the precursor, it has to be produced in a heterologous system. Since both domains contain several cysteines and, due to the known antimicrobial activity of the thionin, we tested the expression of all four Arabidopsis proproteins as fusion proteins. Periplasmic expressi ...[more]

PMID: 23515894

Similar Datasets

RcnB is a periplasmic protein essential for maintaining intracellular Ni and Co concentrations in Escherichia coli.

Project description:Nickel and cobalt are both essential trace elements that are toxic when present in excess. The main resistance mechanism that bacteria use to overcome this toxicity is the efflux of these cations out of the cytoplasm. RND (resistance-nodulation-cell division)- and MFS (major facilitator superfamily)-type efflux systems are known to export either nickel or cobalt. The RcnA efflux pump, which belongs to a unique family, is responsible for the detoxification of Ni and Co in Escherichia coli. In this work, the role of the gene yohN, which is located downstream of rcnA, is investigated. yohN is cotranscribed with rcnA, and its expression is induced by Ni and Co. Surprisingly, in contrast to the effect of deleting rcnA, deletion of yohN conferred enhanced resistance to Ni and Co in E. coli, accompanied by decreased metal accumulation. We show that YohN is localized to the periplasm and does not bind Ni or Co ions directly. Physiological and genetic experiments demonstrate that YohN is not involved in Ni import. YohN is conserved among proteobacteria and belongs to a new family of proteins; consequently, yohN has been renamed rcnB. We show that the enhanced resistance of rcnB mutants to Ni and Co and their decreased Ni and Co intracellular accumulation are linked to the greater efflux of these ions in the absence of rcnB. Taken together, these results suggest that RcnB is required to maintain metal ion homeostasis, in conjunction with the efflux pump RcnA, presumably by modulating RcnA-mediated export of Ni and Co to avoid excess efflux of Ni and Co ions via an unknown novel mechanism.

| S-EPMC3147525 | biostudies-literature

Interaction of Periplasmic Fab Production and Intracellular Redox Balance in <i>Escherichia coli</i> Affects Product Yield.

Project description:Antibody fragments such as Fab's require the formation of disulfide bonds to achieve a proper folding state. During their recombinant, periplasmic expression in Escherichia coli, oxidative folding is mediated by the DsbA/DsbB system in concert with ubiquinone. Thereby, overexpression of Fab's is linked to the respiratory chain, which is not only immensely important for the cell's energy household but also known as a major source of reactive oxygen species. However, the effects of an increased oxidative folding demand and the consequently required electron flux via ubiquinone on the host cell have not been characterized so far. Here, we show that Fab expression in E. coli BL21(DE3) interfered with the intracellular redox balance, thereby negatively impacting host cell performance. Production of four different model Fab's in lab-scale fed-batch cultivations led to increased oxygen consumption rates and strong cell lysis. An RNA sequencing analysis revealed transcription activation of the oxidative stress-responsive soxS gene in the Fab-producing strains. We attributed this to the accumulation of intracellular superoxide, which was measured using flow cytometry. An exogenously supplemented ubiquinone analogue improved Fab yields up to 82%, indicating that partitioning of the quinone pool between aerobic respiration and oxidative folding limited ubiquinone availability and hence disulfide bond formation capacity. Combined, our results provide a more in-depth understanding of the profound effects that periplasmic Fab expression and in particular disulfide bond formation has on the host cell. Thereby, we show new possibilities to elaborate cell engineering and process strategies for improved host cell fitness and process outcome.

| S-EPMC8859853 | biostudies-literature

Pectocin M1 (PcaM1) Inhibits Escherichia coli Cell Growth and Peptidoglycan Biosynthesis through Periplasmic Expression.

Project description:Colicins are bacterial toxins produced by some Escherichia coli strains. They exhibit either enzymatic or pore-forming activity towards a very limited number of bacterial species, due to the high specificity of their reception and translocation systems. Yet, we succeeded in making the colicin M homologue from Pectobacterium carotovorum, pectocin M1 (PcaM1), capable of inhibiting E. coli cell growth by bypassing these reception and translocation steps. This goal was achieved through periplasmic expression of this pectocin. Indeed, when appropriately addressed to the periplasm of E. coli, this pectocin could exert its deleterious effects, i.e., the enzymatic degradation of the peptidoglycan lipid II precursor, which resulted in the arrest of the biosynthesis of this essential cell wall polymer, dramatic morphological changes and, ultimately, cell lysis. This result leads to the conclusion that colicin M and its various orthologues constitute powerful antibacterial molecules able to kill any kind of bacterium, once they can reach their lipid II target. They thus have to be seriously considered as promising alternatives to antibiotics.

| S-EPMC5187517 | biostudies-literature

Directed evolution for soluble and active periplasmic expression of bovine enterokinase in Escherichia coli.

Project description:Bovine enterokinase light chain (EKL) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However, recombinant expression in Escherichia coli produces insoluble inclusion bodies, requiring solubilisation, refolding, and autocatalytic activation to recover functional enzyme. Error-prone PCR and DNA shuffling of the EKL gene, T7 promoter, lac operon, ribosome binding site, and pelB leader sequence, yielded 321 unique variants after screening ~ 6500 colonies. The best variants had > 11,000-fold increased total activity in lysates, producing soluble enzyme that no longer needed refolding. Further characterisation identified the factors that improved total activity from an inactive and insoluble starting point. Stability was a major factor, whereby melting temperatures > 48.4 °C enabled good expression at 37 °C. Variants generally did not alter catalytic efficiency as measured by kcat/Km, which improved for only one variant. Codon optimisation improved the total activity in lysates produced at 37 °C. However, non-optimised codons and expression at 30 °C gave the highest activity through improved protein quality, with increased kcat and Tm values. The 321 variants were statistically analysed and mapped to protein structure. Mutations detrimental to total activity and stability clustered around the active site. By contrast, variants with increased total activity tended to combine stabilising mutations that did not disrupt the active site.

| S-EPMC9587228 | biostudies-literature

The Cytoplasmic and Periplasmic Expression Levels and Folding of Organophosphorus Hydrolase Enzyme in Escherichia coli.

Project description:BackgroundOrganophosphorus hydrolase (OPH) is a type of organophosphate-degrading enzyme which is widely used in the bioremediation process.ObjectivesIn this study, the periplasmic and cytoplasmic productions and the activity of recombinant OPH in Escherichia coli were investigated and compared using two pET systems (pET21a and pET26b).Materials and methodsThe sequence encoding the opd gene was synthesized and expressed in the form of inclusion body using pET21a-opd and in the periplasmic space in pET26b-opd.ResultsSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed a band of about 37 kDa with a maximum expression level at 30°C from pET21a-opd.However, the obtained results of the periplasmic space extraction of OPH (pET26b-opd) showed a very weak band, while the cytoplasmic expression of OPH (pET21a-opd) produced a strong protein band.ConclusionsThe activities studied by the production of PNP were determined by following the increase at 410 nm. The maximum PNP was produced at 30°C with an optical density of 10.62 in the presence of cytoplasmic expression of OPH (pET21a-opd). Consequently, our results suggest cytoplasmic expression system as an appropriate candidate with a high amount of OPH in spite of inclusion body formation, which needs an additional refolding step.

| S-EPMC4746795 | biostudies-literature

The periplasmic protein MppA requires an additional mutated locus to repress marA expression in Escherichia coli.

Project description:Escherichia coli strain TP985, which has an insertional mutation in the gene for the periplasmic murein tripeptide binding protein MppA, was previously reported to overproduce MarA and exhibit a multiple-antibiotic resistance (Mar) phenotype (H. Li and J. T. Park, J. Bacteriol. 181:4842-4847, 1999). We found that TP985 contained a previously unrecognized marR mutation which was responsible for the Mar phenotype. Transduction of the mppA mutation from TP985 to another wild-type strain did not affect antibiotic susceptibility. Overproduction of MppA repressed marA transcription in TP985 but not in other mppA or marR mutants. Therefore, TP985 contains an additional unknown mutation(s) that facilitates the repression of marA expression by MppA.

| S-EPMC142866 | biostudies-literature

Intracellular expression of the plasmid-encoded toxin from enteroaggregative Escherichia coli.

Project description:The plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a serine protease autotransporter that acts as an enterotoxin and cytotoxin. When applied to epithelial cells in culture, purified toxin induces cell elongation and rounding, followed by exfoliation of cells from the substratum. These effects are accompanied by loss of actin stress fibers and electrophysiologic changes. Although it has been hypothesized that Pet has an intracellular site of action, evidence for this is indirect. In addition, Pet has recently been shown to cleave spectrin in vitro and in vivo. If Pet requires intracellular localization to execute its toxic effects, then intracellular expression of the protein could induce cytopathic effects similar to those observed when the toxin is applied to the cell surface. To test this hypothesis, we expressed the mature Pet toxin (comprising only the passenger domain of the Pet precursor) in the cytoplasm of HEp-2 cells by using mammalian expression vectors. Separately, we expressed the Pet passenger domain mutated at the catalytic serine (PetS260A), a construct that has been reported to lack toxic effects. Forty-eight hours after transient transfection of pcDNA3.1-pet in HEp-2 cells, we observed cell elongation and other morphological changes similar to those induced by applied toxin. Cells transfected with pcDNA3.1 vector alone appeared normal, while cells expressing the PetS260A mutant displayed similar (though less pronounced) changes compared with those in cells expressing pcDNA3.1-pet. Notably, intracellular expression of Pet was accompanied by condensation of the spectrin cytoskeleton. These studies corroborate an intracellular site of action for the Pet toxin, further implicate a role for spectrin in Pet intoxication, and provide a powerful tool for Pet structure and function analyses.

| S-EPMC187336 | biostudies-literature

The thionin family of antimicrobial peptides.

Project description:Thionins are antimicrobial peptides found only in plants. They are first produced as preproproteins and then processed to yield the usually 5 kDa, basic thionin peptide with three or four disulfide bridges. So far, thionins had only been found in some plant families of angiosperms. The One Thousand Plant Transcriptomes Initiative (1KP project) has sequenced the transcriptomes of more than 1000 plant species. We have used these data to search for new thionin sequences which gave 225 hits. After removing doublets these resulted in 133 new thionins. No sequences were found in algae and mosses. The phylogenetically earliest hits were from Selaginella species and from conifers. Many hits were from angiosperm plant families which were previously not known to contain thionins. A large gene family for thionins was found in Papaver. We isolated a genomic clone from Papaver somniferum which confirmed the general genomic structure with two small introns within the acidic domain. We also expressed the thionin encoded by the genomic clone and found that it had antimicrobial activity in vitro, especially against fungi. Previously, we had grouped thionins into four classes. The new data reported here led us to revise this classification. We now recognize only class 1 thionins with eight cysteine residues and class 2 thionins with six cysteine residues. The different variants that we found (and also previously known variants) can all be traced back to one of these two classes. Some of the variants had an uneven number of cysteine residues and it is not clear at the moment what that means for their threedimensional structure.

| S-EPMC8279376 | biostudies-literature

Protein fusion tags for efficient expression and purification of recombinant proteins in the periplasmic space of E. coli.

Project description:Disulfide bonds occurred in majority of secreted protein. Formation of correct disulfide bonds are must for achieving native conformation, solubility and activity. Production of recombinant proteins containing disulfide bond for therapeutic, diagnostic and various other purposes is a challenging task of research. Production of such proteins in the reducing cytosolic compartment of E. coli usually ends up in inclusion bodies formation. Refolding of inclusion bodies can be difficult, time and labor consuming and uneconomical. Translocation of these proteins into the oxidative periplasmic compartment provides correct environment to undergo proper disulfide bonds formation and thus achieving native conformation. However, not all proteins can be efficiently translocated to the periplasm with the help of bacterial signal peptides. Therefore, fusion to a small well-folded and stable periplasmic protein is more promising for periplasmic production of disulfide bonded proteins. In the past decades, several full-length proteins or domains were used for enhancing translocation and solubility. Here, protein fusion tags that significantly increase the yields of target proteins in the periplasmic space are reviewed.

| S-EPMC4742420 | biostudies-literature

Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries.

Project description:Anchored periplasmic expression (APEx) is a technology for the isolation of ligand-binding proteins from combinatorial libraries anchored on the periplasmic face of the inner membrane of Escherichia coli. After disruption of the outer membrane by Tris-EDTA-lysozyme, the inner-membrane-anchored proteins readily bind fluorescently labeled ligands as large as 240 kDa. Fluorescently labeled cells are isolated by flow cytometry, and the DNA of isolated clones is rescued by PCR. By using two rounds of APEx, the affinity of a neutralizing antibody to the Bacillus anthracis protective antigen was improved >200-fold, exhibiting a final K(D) of 21 pM. This approach has several technical advantages compared with previous library screening technologies, including the unique ability to screen for ligand-binding proteins that bind endogenously expressed ligands fused to a short-lived GFP. Further, APEx is able to display proteins either as an N-terminal fusion to a six-residue sequence derived from the native E. coli lipoprotein NlpA, or as a C-terminal fusion to the phage gene three minor coat protein of M13. The latter fusions allow hybrid phage display/APEx strategies without the need for further subcloning.

| S-EPMC438952 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data