Improved Strains and Plasmid Vectors for Conditional Overexpression of His-Tagged Proteins in
            <i>Haloferax volcanii</i>

Allers, Thorsten; Barak, S.; Liddell, Susan; Wardell, Kayleigh; Mevarech, Moshe

doi:10.1128/aem.02670-09

Cited by 188 publications

(220 citation statements)

References 41 publications

Supporting

Mentioning

220

Contrasting

Order By: Relevance

“…These large differences presumably explain why no recombinant expression of the alcohol dehydrogenase ADH/D1 was observed in E. coli. The engineered H. volcanii expression system (Allers 2010 ;Allers et al 2004Allers et al , 2010 was capable of producing moderate amounts of protein in shaker-flask cultures, the essential step for the biotechnological use of halophilic enzymes remains a large-scale production in bioreactors. The method established in this study facilitates straightforward batch cultivation of H. volcanii H1895 in a stirred-tank bioreactor using optimised settings and media.…”

Section: Discussionmentioning

confidence: 99%

“…To date, several strategies have been applied to engineer H. volcanii for improving heterologous gene expression resulting in the H1424 strain (Allers 2010 ;Allers et al 2010 ). The corresponding plasmid to this strain is pTA963 with the inducible tryptophanase promoter p.tna, allowing the induction of intracellular protein overexpression by L-tryptophan (Allers 2010 ;Allers et al 2004Allers et al , 2010Bitan-Banin et al 2003 ;Large et al 2007 ).…”

Section: Introductionmentioning

confidence: 99%

“…The corresponding plasmid to this strain is pTA963 with the inducible tryptophanase promoter p.tna, allowing the induction of intracellular protein overexpression by L-tryptophan (Allers 2010 ;Allers et al 2004Allers et al , 2010Bitan-Banin et al 2003 ;Large et al 2007 ). The expression vector is based on the pHV2 origin which maintains the plasmid at a copy number of approximately 6 per genome equivalent (Allers 2010 ).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor

Strillinger

Grötzinger

Allers

et al. 2015

Appl Microbiol Biotechnol

Self Cite

View full text Add to dashboard Cite

The success of biotechnological processes is based on the availability of efficient and highly specific biocatalysts, which can satisfy industrial demands. Extreme and remote environments like the deep brine pools of the Red Sea represent highly interesting habitats for the discovery of novel halophilic and thermophilic enzymes. Haloferax volcanii constitutes a suitable expression system for halophilic enzymes obtained from such brine pools. We developed a batch process for the cultivation of H. volcanii H1895 in controlled stirred-tank bioreactors utilising knockouts of components of the flagella assembly system. The standard medium Hv-YPC was supplemented to reach a higher cell density. Without protein expression, cell dry weight reaches 10 g L . Two halophilic alcohol dehydrogenases were expressed under the control of the tryptophanase promoter p.tna with 16.8 and 3.2 mg g , respectively, at a maximum cell dry weight of 6.5 g L . Protein expression was induced by the addition of L-tryptophan. Investigation of various expression strategies leads to an optimised two-step induction protocol introducing 6 mM L-tryptophan at an OD of 0.4 followed by incubation for 16 h and a second induction step with 3 mM L-tryptophan followed by a final incubation time of 4 h. Compared with the uncontrolled shaker-flask cultivations used until date, dry cell mass concentrations were improved by a factor of more than 5 and cell-specific enzyme activities showed an up to 28-fold increased yield of the heterologous proteins.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor

Strillinger

Grötzinger

Allers

et al. 2015

Appl Microbiol Biotechnol

Self Cite

View full text Add to dashboard Cite

show abstract

“…By contrast, as found in type-II AAA+ enzymes, the homohexameric Cdc48 motor in the recently discovered archaeal Cdc48·20S proteasome contains two AAA+ modules (7,8). Cdc48 is essential in archaea and eukaryotes, and structural studies of a mouse ortholog have shown that its AAA+ modules are organized into discrete D1 and D2 rings (9)(10)(11). Cdc48 also contains a family-specific N domain, as well as a flexible C-terminal sequence, including an HbYX (hydrophobic, tyrosine, any residue) tripeptide important for 20S binding, which is similar to motifs in Mpa/Arc, Pan, and Rpt 1-6 (8).…”

mentioning

confidence: 99%

Architecture and assembly of the archaeal Cdc48⋅20S proteasome

Barthelme¹,

Chen²,

Grabenstatter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

ATP-dependent proteases maintain protein quality control and regulate diverse intracellular functions. Proteasomes are primarily responsible for these tasks in the archaeal and eukaryotic domains of life. Even the simplest of these proteases function as large complexes, consisting of the 20S peptidase, a barrel-like structure composed of four heptameric rings, and one or two AAA+ (ATPase associated with a variety of cellular activities) ring hexamers, which use cycles of ATP binding and hydrolysis to unfold and translocate substrates into the 20S proteolytic chamber. Understanding how the AAA+ and 20S components of these enzymes interact and collaborate to execute protein degradation is important, but the highly dynamic nature of prokaryotic proteasomes has hampered structural characterization. Here, we use electron microscopy to determine the architecture of an archaeal Cdc48·20S proteasome, which we stabilized by site-specific cross-linking. This complex displays coaxial alignment of Cdc48 and 20S and is enzymatically active, demonstrating that AAA+ unfoldase wobbling with respect to 20S is not required for function. In the complex, the N-terminal domain of Cdc48, which regulates ATP hydrolysis and degradation, packs against the D1 ring of Cdc48 in a coplanar fashion, constraining mechanisms by which the N-terminal domain alters 20S affinity and degradation activity.AAA+ protease | dynamic wobbling model | p97/VCP P roteasomes are large macromolecular complexes that degrade misfolded or damaged proteins to maintain cellular homeostasis and quality control in all domains of life. In addition, selective proteasomal turnover of regulatory proteins is often a critical element of signaling cascades that allow cells to respond to changing conditions and environmental stress (1, 2). Proteasomes consist of the self-compartmentalized 20S peptidase and one or two AAA+ (ATPase associated with a variety of cellular activities) family ring hexamers. The α 7 β 7 β 7 α 7 ring topology of the 20S enzyme ensures that the proteolytic active sites, which reside in the β-subunits, are sequestered and can only cleave substrates that enter the chamber through narrow axial pores formed by the α-subunits (3). As a consequence, ATP-dependent unfolding of natively folded protein substrates by the AAA+ ring and subsequent polypeptide translocation through a narrow axial channel and into the 20S chamber are required for degradation (4).Although the 20S peptidase is the degradation module in all proteasomes, the associated AAA+ unfolding machines differ. For example, homohexamers of Mpa/Arc in actinobacteria and PAN in archaea serve as proteasomal motors, whereas a heterohexameric Rpt 1-6 ring in the 19S particle is the motor of the eukaryotic 26S proteasome (2, 5). Characteristic of type-I AAA+ enzymes, Mpa/Arc, PAN, and Rpt 1-6 subunits contain a single AAA+ module for ATP binding and hydrolysis (6). By contrast, as found in type-II AAA+ enzymes, the homohexameric Cdc48 motor in the recently discovered archaeal Cdc48·20S proteasome cont...

show abstract

“…Notably, deletion mutants can be constructed very easily, 32,33 it can be grown in microtiter plates enabling phenotyping of mutants, 31 transcriptome analysis using DNA microarrays has been established, 34,35 proteome analysis is routinely performed, 36,37 and many molecular genetic tools are available. [38][39][40] Here we report the application of two bioinformatic approaches to predict sRNA genes in the genome in H. volcanii by comparative genomics of intergenic regions. In addition, (differential) expression of the predicted putative sRNA genes was characterized by DNA microarray analysis and by comparison with the results of high throughput sequencing performed in a parallel study.…”

Section: Introductionmentioning

confidence: 99%

Bioinformatic prediction and experimental verification of sRNAs in the haloarchaeonHaloferax volcanii

Babski¹,

Tjaden²,

Voss³

et al. 2011

RNA Biology

View full text Add to dashboard Cite

Improved Strains and Plasmid Vectors for Conditional Overexpression of His-Tagged Proteins in Haloferax volcanii

Cited by 188 publications

References 41 publications

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor

Architecture and assembly of the archaeal Cdc48⋅20S proteasome

Bioinformatic prediction and experimental verification of sRNAs in the haloarchaeonHaloferax volcanii

Contact Info

Product

Resources

About