Cloning and expression of Vibrio cholerae dsbA, a gene encoding a periplasmic protein disulphide isomerase

1995 ◽  
Vol 23 (1) ◽  
pp. 64S-64S ◽  
Author(s):  
EDWARD D. LOWE ◽  
ROBERT B. FREEDMAN ◽  
TIMOTHY R. HIRST ◽  
PETER T. BARTH
Keyword(s):  
Vibrio Cholerae ◽  
Cloning And Expression ◽  
Periplasmic Protein ◽  
Gene Encoding ◽  
Protein Disulphide Isomerase

Cloning and Expression of the xynA1 Gene Encoding a Xylanase of the GH10 Group in Caulobacter crescentus

2015 ◽  
Vol 175 (8) ◽  
pp. 3915-3929 ◽  
Author(s):  
Luciana Graciano ◽  
Juliana Moço Corrêa ◽  
Fabíola Giovanna Nesello Vieira ◽  
Adilson Bosetto ◽  
Eduardo Alexandre Loth ◽  
...  
Keyword(s):  
Caulobacter Crescentus ◽  
Cloning And Expression ◽  
Gene Encoding

scholarly journals Baculovirus expression of two protein disulphide isomerase isoforms from Caenorhabditis elegans and characterization of prolyl 4-hydroxylases containing one of these polypeptides as their β subunit

1996 ◽  
Vol 317 (3) ◽  
pp. 721-729 ◽  
Author(s):  
Johanna VEIJOLA ◽  
Pia ANNUNEN ◽  
Peppi KOIVUNEN ◽  
Antony P. PAGE ◽  
Taina PIHLAJANIEMI ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Cloning And Expression ◽  
Β Subunit ◽  
Α Subunit ◽  
Protein Disulphide Isomerase ◽  
C Elegans ◽  
Baculovirus Expression ◽  
Expression Studies ◽  
Α Subunits

Protein disulphide isomerase (PDI; EC 5.3.4.1) is a multifunctional polypeptide that is identical to the β subunit of prolyl 4-hydroxylases. We report here on the cloning and expression of the Caenorhabditis elegans PDI/β polypeptide and its isoform. The overall amino acid sequence identity and similarity between the processed human and C. elegans PDI/β polypeptides are 61% and 85% respectively, and those between the C. elegans PDI/β polypeptide and the PDI isoform 46% and 73%. The isoform differs from the PDI/β and ERp60 polypeptides in that its N-terminal thioredoxin-like domain has an unusual catalytic site sequence -CVHC-. Expression studies in insect cells demonstrated that the C. elegans PDI/β polypeptide forms an active prolyl 4-hydroxylase α2β2 tetramer with the human α subunit and an αβ dimer with the C. elegans α subunit, whereas the C. elegans PDI isoform formed no prolyl 4-hydroxylase with either α subunit. Removal of the 32-residue C-terminal extension from the C. elegans α subunit totally eliminated αβ dimer formation. The C. elegans PDI/β polypeptide formed less prolyl 4-hydroxylase with both the human and C. elegans α subunits than did the human PDI/β polypeptide, being particularly ineffective with the C. elegans α subunit. Experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human and C. elegans PDI/β polypeptides indicated that differences in the C-terminal region are one reason, but not the only one, for the differences in prolyl 4-hydroxylase formation between the human and C. elegans PDI/β polypeptides. The catalytic properties of the C. elegans prolyl 4-hydroxylase αβ dimer were very similar to those of the vertebrate type II prolyl 4-hydroxylase tetramer, including the Km for the hydroxylation of long polypeptide substrates.

scholarly journals BrucellaPeriplasmic Protein EipB Is a Molecular Determinant of Cell Envelope Integrity and Virulence

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Julien Herrou ◽  
Jonathan W. Willett ◽  
Aretha Fiebig ◽  
Daniel M. Czyż ◽  
Jason X. Cheng ◽  
...  
Keyword(s):  
Cell Envelope ◽  
Mammalian Host ◽  
Periplasmic Protein ◽  
Periplasmic Space ◽  
Gene Encoding ◽  
Tetratricopeptide Repeats ◽  
Content Type ◽  
Family Protein ◽  
Synthetically Lethal

ABSTRACTThe Gram-negative cell envelope is a remarkable structure with core components that include an inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. Multiple molecular systems function to maintain integrity of this essential barrier between the interior of the cell and its surrounding environment. We show that a conserved DUF1849 family protein, EipB, is secreted to the periplasmic space ofBrucellaspecies, a monophyletic group of intracellular pathogens. In the periplasm, EipB folds into an unusual 14-stranded β-spiral structure that resembles the LolA and LolB lipoprotein delivery system, though the overall fold of EipB is distinct from LolA/LolB. Deletion ofeipBresults in defects inBrucellacell envelope integrityin vitroand in maintenance of spleen colonization in a mouse model ofBrucella abortusinfection. Transposon disruption ofttpA, which encodes a periplasmic protein containing tetratricopeptide repeats, is synthetically lethal witheipBdeletion.ttpAis a reported virulence determinant inBrucella, and our studies ofttpAdeletion and overexpression strains provide evidence that this gene also contributes to cell envelope function. We conclude thateipBandttpAfunction in theBrucellaperiplasmic space to maintain cell envelope integrity, which facilitates survival in a mammalian host.IMPORTANCEBrucellaspecies cause brucellosis, a global zoonosis. A gene encoding a conserved DUF1849-family protein, which we have named EipB, is present in all sequencedBrucellaand several other genera in the classAlphaproteobacteria. The manuscript provides the first functional and structural characterization of a DUF1849 protein. We show that EipB is secreted to the periplasm where it forms a spiral-shaped antiparallel β protein that is a determinant of cell envelope integrityin vitroand virulence in an animal model of disease.eipBgenetically interacts withttpA, which also encodes a periplasmic protein. We propose that EipB and TtpA function as part of a system required for cell envelope homeostasis in selectAlphaproteobacteria.

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