scholarly journals Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization

1995 ◽  
Vol 310 (2) ◽  
pp. 605-614 ◽  
Author(s):  
M J Stone ◽  
W Ruf ◽  
D J Miles ◽  
T S Edgington ◽  
P E Wright
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Tissue Factor ◽  
Inclusion Bodies ◽  
Factor Viia ◽  
Factor X ◽  
Wild Type ◽  
Proteolytic Activation ◽  
E Coli

Tissue factor (TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades. The abilities of TF to associate with Factor VIIa and Factor X in a ternary complex and to enable proteolytic activation of Factor X by Factor VIIa reside in the extracellular domain of TF. We describe the expression of the surface domain of TF (truncated TF, tTF) in both Saccharomyces cerevisiae and Escherichia coli and the biochemical and physical characterization of the recombinant proteins. Wild-type tTF and several glycosylation-site mutants were secreted efficiently by S. cerevisiae under the control of the yeast prepro-alpha-signal sequence; the T13A,N137D double mutant was the most homogeneous variant expressed in milligram quantities. Wild-type tTF was expressed in a non-native state in E. coli inclusion bodies as a fusion protein with a poly(His) leader. The fusion protein could be fully renatured and the leader removed by proteolysis with thrombin; the correct molecular mass (24,729 Da) of the purified protein was confirmed by electrospray mass spectrometry. Recombinant tTFs from yeast, E. coli and Chinese hamster ovary cells were identical in their abilities to bind Factor VIIa, to enhance the catalytic activity of Factor VIIa and to enhance the proteolytic activation of Factor X by Factor VIIa. Furthermore, CD, fluorescence emission and NMR spectra of the yeast and E. coli proteins indicated that these proteins are essentially identical structurally.

Activation of Factor X by Factor VIIa Complexed with Human-mouse Tissue Factor Chimeras Requires Human Exon 3

1996 ◽  
Vol 76 (03) ◽  
pp. 361-368 ◽  
Author(s):  
Carrie H Fang ◽  
T-C Lin ◽  
Arabinda Guha ◽  
Yale Nemerson ◽  
William H Konigsberg
Keyword(s):  
Tissue Factor ◽  
Human Factor ◽  
Factor Viia ◽  
Mouse Tissue ◽  
Factor X ◽  
Lower Affinity ◽  
E Coli ◽  
Sequence Elements ◽  
Specific Activities ◽  
Human And Mouse

SummaryIn an attempt to define sequence elements in human and mouse tissue factor (TF) that are responsible for the species specificity observed in their interaction with human factor VIIa (HVIIa), we constructed human-mouse chimeric TF cDNAs, inserted them into plasmid vectors, and induced their expression in E.coli. Assays for procoagulant activity were carried out with the resulting E. coli lysates using (HVIIa) human and mouse (MVIIa). The ratio of the procoagulant activities, HVIIa/MVIIa, revealed that human TF exon 3 was essential for activity when the TF:VIIa complex was formed with HVIIa. By ligating the maltose binding protein (MBP) gene to TF cDNAs it was possible to construct, express and purify MBP-TF chimeras as well as to estimate their specific activities. With selected MBP-TF chimeras and HVIIa we determined kinetic parameters for the activation of human factor X. Replacement of exon 3 in human TF cDNA with the corresponding exon from mouse TF cDNA resulted in both lower affinity for HVIIa and failure to convert bound HVIIa into a potent protease

scholarly journals Characterization of the inhibition of tissue factor in serum

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 150-155 ◽  
Author(s):  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Factor Vii ◽  
Factor X ◽  
Human Hepatoma Cells ◽  
Proteolytic Activation

Tissue factor (TF) is a lipoprotein cofactor that markedly enhances the proteolytic activation of factors IX and X by factor VIIa. The functional activity of TF is inhibited by serum in a time- and temperature-dependent fashion. The inhibitory effect is also dependent on the presence of calcium ions and can be reversed by calcium chelation (EDTA) and dilution, thus excluding direct proteolytic destruction of TF as the mechanism for inhibition. Using crude TF, serum immunodepleted of factor VII, and serum depleted of the vitamin K- dependent coagulation factors by BaSO4 absorption, it is shown that TF factor inhibition requires the presence of VII(a), X(a), and an additional moiety contained in barium-absorbed serum. When each of the other required components were at saturating concentrations, half- maximal inhibition of TF occurred in reaction mixtures containing 2% (vol/vol) of TF at a factor VII(a) concentration of 4 ng/mL (80 pmol/L), a factor X concentration of 50 ng/mL (850 pmol/L), and a concentration of barium-absorbed serum of 2.5% (vol/vol). Catalytically active factor Xa appeared to be required for the generation of optimal TF inhibition. The results are consistent with the conclusions of Hjort that barium-absorbed serum contains a moiety that inhibits the VIIa- Ca2+-TF complex. The role of factor X(a) in the generation of the inhibitory phenomenon remains to be elucidated. The inhibitor present in serum (plasma) may in part be produced by the liver in vivo since cultured human hepatoma cells (HepG2) secrete this inhibitory activity in vitro.

scholarly journals Influence of mutations in tissue factor on the fine specificity of macromolecular substrate activation

1997 ◽  
Vol 321 (3) ◽  
pp. 787-794 ◽  
Author(s):  
Sabine DITTMAR ◽  
Wolfram RUF ◽  
Thomas S. EDGINGTON
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Factor Ix ◽  
Factor X ◽  
Wild Type ◽  
Binding Studies ◽  
Fine Specificity ◽  
Fibronectin Type Iii

The C-terminal fibronectin-type-III-like module of the tissue factor (TF) extracellular domain plays a requisite role in the activation of macromolecular substrates by factor VIIa (VIIa) in complex with TF. Unlike the mutations Lys165→Ala, Lys166→Ala in TF, which prevent efficient proteolysis of factor X, we found that the coagulant defect of a site-specific Trp158→Arg, Ser160→Gly replacement mutant of TF is largely attributable to the inability of TF to efficiently support the activation of the bound zymogen VII to the active protease VIIa. Binding studies demonstrated comparable affinity of binding of VIIa or VII by wild-type TF and TFR158G160. In comparison with wild-type TF, the catalytic efficiency of factor X activation was reduced 56-fold with TFA165A166 as the cofactor, but only 3.5-fold with TFR158G160. The activation of VII bound to TF by factor Xa or VIIa was reduced 2-fold in the presence of TFR158G160 and 7Ő8-fold with TFA165A166. This suggests that the molecular recognition of VII in complex with TF by the enzymes TFŐVIIa and factor Xa are similar. Generation of factor IXa by TFR158G160ŐVIIa was unaltered, but reduced 2-fold with TFA165A166. In addition, the mutations affected the cleavage of the two scissile bonds of factor IX differently, providing further support for the idea that the cofactor, TF, influences the fine specificity of activation of macromolecular substrates by the TFŐVIIa complex.

scholarly journals Purification and characterization of recombinant pea-seed ferritins expressed in Escherichia coli: influence of N-terminus deletions on protein solubility and core formation in vitro

1995 ◽  
Vol 305 (1) ◽  
pp. 253-261 ◽  
Author(s):  
O Van Wuytswinkel ◽  
G Savino ◽  
J F Briat
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Dna Fragments ◽  
Core Formation ◽  
Wild Type ◽  
E Coli ◽  
N Terminus ◽  
Ferroxidase Activity ◽  
Pea Seed

Plant ferritin subunits are synthesized as precursor molecules; the transit peptide (TP) in their NH2 extremity, responsible for plastid targeting, is cleaved during translocation to this compartment. In addition, the N-terminus of the mature subunit contains a plant-specific sequence named extension peptide (EP) [Ragland, Briat, Gagnon, Laulhère, Massenet, and Theil, E.C. (1990) J. Biol. Chem. 265, 18339-18344], the function of which is unknown. A novel pea-seed ferritin cDNA, with a consensus ferroxidase centre conserved within H-type animal ferritins has been characterized. This pea-seed ferritin cDNA has been engineered using oligonucleotide-directed mutagenesis to produce DNA fragments (1) corresponding to the wild-type (WT) ferritin precursor, (2) with the TP deleted, (3) with both the TP and the plant specific EP sequences deleted and (4) containing the TP but with the EP deleted. These four DNA fragments have been cloned in an Escherichia coli expression vector to produce the corresponding recombinant pea-seed ferritins. Expression at 37 degrees C led to the accumulation of recombinant pea-seed ferritins in inclusion bodies, whatever the construct introduced in E. coli. Expression at 25 degrees C in the presence of sorbitol and betaine allowed soluble proteins to accumulate when constructs with the TP deleted were used; under this condition, E. coli cells transformed with constructs containing the TP were unable to accumulate recombinant protein. Recombinant ferritins purified from inclusion bodies were found to be assembled only when the TP was deleted; however assembled ferritin under this condition had a ferroxidase activity undetectable at acid pH. On the other hand, soluble recombinant ferritins with the TP deleted and expressed at 25 degrees C were purified as 24-mers containing an average of 40-50 iron atoms per molecule. Despite the conservation in the plant ferritin subunit of a consensus ferroxidase centre, the iron uptake activity in vitro at pH 6.8 was found to be lower than that of the recombinant human H-ferritin, though it was much more active than the recombinant human L-ferritin. The recombinant ferritin with both the TP and the EP deleted (r delta TP/EP) assembled correctly as a 24-mer; it has slightly higher ferroxidase activity and decreased solubility compared with the wild-type protein with the TP deleted (r delta TP). In addition, on denaturation by urea followed by renaturation by dialysis the r delta TP/EP protein showed a 25% increase in core-formation in vitro compared with the r delta TP protein.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Expression of the Mitochondrial ADP/ATP Carrier inEscherichia coli

2001 ◽  
Vol 276 (15) ◽  
pp. 11499-11506 ◽  
Author(s):  
Simone Heimpel ◽  
Gabriele Basset ◽  
Sabine Odoy ◽  
Martin Klingenberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Inclusion Bodies ◽  
Positive Charge ◽  
Inescherichia Coli ◽  
Transport Activity ◽  
E Coli ◽  
The Matrix ◽  
A Charge

Previously, the role of residues in the ADP/ATP carrier (AAC) fromSaccharomyces cerevisiaehas been studied by mutagenesis, but the dependence of mitochondrial biogenesis on functional AAC impedes segregation of the mutational effects on transport and biogenesis. Unlike other mitochondrial carriers, expression of the AAC from yeast or mammalians inEscherichia coliencountered difficulties because of disparate codon usage. Here we introduce the AAC fromNeurospora crassainE. coli, where it is accumulated in inclusion bodies and establish the reconstitution conditions. AAC expressed with heat shock vector gave higher activity than with pET-3a. Transport activity was absolutely dependent on cardiolipin. The 10 single mutations of intrahelical positive residues and of the matrix repeat (+X+) motif resulted in lower activity, except of R245A. R143A had decreased sensitivity toward carboxyatractylate. The ATP-linked exchange is generally more affected than ADP exchange. This reflects a charge network that propagates positive charge defects to ATP4−more strongly than to ADP3−transport. Comparison to the homologous mutants of yeast AAC2 permits attribution of the roles of these residues more to ADP/ATP transport or to AAC import into mitochondria.

Blockade of tissue factor-factor X binding attenuates sepsis-induced respiratory and renal failure

2006 ◽  
Vol 290 (1) ◽  
pp. L21-L31 ◽  
Author(s):  
Karen E. Welty-Wolf ◽  
Martha S. Carraway ◽  
Thomas L. Ortel ◽  
Andrew J. Ghio ◽  
Steven Idell ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Lung Compliance ◽  
Protective Effects ◽  
Factor X ◽  
Fibrin Deposition ◽  
Antibody Treatment ◽  
E Coli ◽  
Colony Forming Units ◽  
Factor Expression

Tissue factor expression in sepsis activates coagulation in the lung, which potentiates inflammation and leads to fibrin deposition. We hypothesized that blockade of factor X binding to the tissue factor-factor VIIa complex would prevent sepsis-induced damage to the lungs and other organs. Acute lung injury was produced in 15 adult baboons primed with killed Escherichia coli [1 × 109colony-forming units (CFU)/kg], and then 12 h later, they were given 1 × 1010CFU/kg live E. coli by infusion. Two hours after live E. coli, animals received antibiotics with or without monoclonal antibody to tissue factor intravenously to block tissue factor-factor X binding. The animals were monitored physiologically for 34 h before being killed and their tissue harvested. The antibody treatment attenuated abnormalities in gas exchange and lung compliance, preserved renal function, and prevented tissue neutrophil influx and bowel edema relative to antibiotics alone (all P < 0.05). It also attenuated fibrinogen depletion ( P < 0.01) and decreased proinflammatory cytokines, e.g., IL-6 and -8 ( P < 0.01), in systemic and alveolar compartments. Similar protective effects of the antibody on IL-6 and -8 expression and permeability were found in lipopolysaccharide-stimulated endothelial cells. Blockade of factor X binding to the tissue factor-factor VIIa complex attenuates lung and organ injuries in established E. coli sepsis by attenuating the neutrophilic response and inflammatory pathways.

Coagulation factor III (tissue factor) interaction with phospholipid vesicles induced by cadmium: characterization of the reconstituted protein-membrane complex

1981 ◽  
Vol 1 (3) ◽  
pp. 197-205 ◽  
Author(s):  
Steven D. Carson ◽  
William H. Konigsberg
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Gel Filtration ◽  
Coagulation Factor ◽  
Bovine Brain ◽  
Factor Ix ◽  
Phospholipid Vesicles ◽  
Factor X ◽  
Lipid Complex ◽  
Proteolytic Activation

Coagulation factor III (tissue factor) is a membrane glycoprotein which serves as a cofactor in the proteolytic activation of factor X and factor IX by factor VIIa. Mixing of human placental factor III apoprotein with vesicles of bovine brain phospholipids does not produce significant reconstitution of factor III activity, but, when the mixture of apoprotein and vesicles is made 5 mM with CdCI2, the apoprotein is incorporated into the vesicles. Ultracentrifugation on sucrose density gradients demonstrated that the active factor III-lipid complex formed by reconstitution with vesicles had a density indistinguishable from that of the complex formed by detergent dialysis. Vesicles isolated after centrifugation were shown to range in diameter from 20 nm to over 100 nm using the electron microscope. Gel filtration showed that factor-III activity was associated with all size-classes of vesicles. The presence of factor III activity in the smaltest vesicles argues for a specific cadmium-mediated reconstitution of the apoprotein with phospholipid vesicles.

Expression of a Fusion Protein of Human Proinsulin with Glutathione-S-Transferase in Escherichia coli

2014 ◽  
Vol 998-999 ◽  
pp. 248-251
Author(s):  
Zhi Xin Di ◽  
Jian Zhong Ma ◽  
Yong Gang Wang
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Glutathione S Transferase ◽  
E Coli ◽  
Human Proinsulin ◽  
Sds Page ◽  
Dna Fragment ◽  
Sequence Encoding

A DNA sequence encoding for the human proinsulin was designed according to the codon bias of Escherichia coli and then chemically synthesized. The synthesized DNA fragment was subcloned into pGEX-3X for expression in E. coli BL21 (DE3) and E. coli BL21 Star (DE3), respectively. Conditions for the highest expression of the GST-proinsulin fusion proteins were optimized. These conditions are that cells of E. coli BL21 star (DE3) are incubated in 100mL of the LB medium with 2 mmol/L IPTG and 60μ?g/mL ampicillin at 26oCfor 4h. After disrupted E. coli cells with ultrasonication, inclusion bodies were precipitated from cell lysis and washed. Fusion proteins from the inclusion bodies were redissolved in 8mmol/L of urea. After dialysed in purified water, fusion proteins were analysed by SDS-PAGE. The purity of the fusion protein is about 80.5% in total. The fusion protein from SDS-PAGE was further identified by mass/mass spectrum. GST in the dyad protein is confirmed by the 9 matched sequences. However, the left part is proved a polypeptide of which is completely different from the human proinsulin.

scholarly journals Characterization of the inhibition of tissue factor in serum

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 150-155 ◽  
Author(s):  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Factor Vii ◽  
Factor X ◽  
Human Hepatoma Cells ◽  
Proteolytic Activation

Abstract Tissue factor (TF) is a lipoprotein cofactor that markedly enhances the proteolytic activation of factors IX and X by factor VIIa. The functional activity of TF is inhibited by serum in a time- and temperature-dependent fashion. The inhibitory effect is also dependent on the presence of calcium ions and can be reversed by calcium chelation (EDTA) and dilution, thus excluding direct proteolytic destruction of TF as the mechanism for inhibition. Using crude TF, serum immunodepleted of factor VII, and serum depleted of the vitamin K- dependent coagulation factors by BaSO4 absorption, it is shown that TF factor inhibition requires the presence of VII(a), X(a), and an additional moiety contained in barium-absorbed serum. When each of the other required components were at saturating concentrations, half- maximal inhibition of TF occurred in reaction mixtures containing 2% (vol/vol) of TF at a factor VII(a) concentration of 4 ng/mL (80 pmol/L), a factor X concentration of 50 ng/mL (850 pmol/L), and a concentration of barium-absorbed serum of 2.5% (vol/vol). Catalytically active factor Xa appeared to be required for the generation of optimal TF inhibition. The results are consistent with the conclusions of Hjort that barium-absorbed serum contains a moiety that inhibits the VIIa- Ca2+-TF complex. The role of factor X(a) in the generation of the inhibitory phenomenon remains to be elucidated. The inhibitor present in serum (plasma) may in part be produced by the liver in vivo since cultured human hepatoma cells (HepG2) secrete this inhibitory activity in vitro.

Factors Affecting the lnteraction of Tissue Factor/Factor Vll with Factor X in a Heterogeneous Tubular Reactor

1991 ◽  
Vol 65 (02) ◽  
pp. 139-143 ◽  
Author(s):  
Cynthia H Gemmell ◽  
Vincet T Turitto ◽  
Yale Nemerson
Keyword(s):  
Steady State ◽  
Tissue Factor ◽  
Factor Viia ◽  
Transmembrane Protein ◽  
Strong Association ◽  
Tubular Reactor ◽  
Factor Xa ◽  
Phospholipid Bilayer ◽  
Factor Vii ◽  
Factor X

SummaryA novel reactor recently described for studying phospholipiddependent blood coagulation reactions under flow conditions similar to those occurring in the vasculature has been further charactenzed. The reactor is a capitlary whose inner wall is coated with a stable phospholipid bilayer (or two bilayers) containing tissue factor, a transmembrane protein that is required for the enzymatic activation of factor X by factor VIIa. Perfusion of the capillary at wall shear rates ranging from 25 s−1 to 1,200 s−1 with purified bovine factors X and VIIa led to steady state factor Xa levels at the outlet. Assay were performed using a chromogenic substrate, SpectrozymeTMFXa, or by using a radiometric technique. In the absence of Ca2+ or factor VIIa there was no product formation. No difference was noted in the levels of factor Xa achieved when non-activated factor VII was perfused. Once steady state was achieved further factor Xa production continued in the absence of factor VIIa implying a very strong association of factor VIIa with the tissue factor in the phospholipid membrane. In agreement with static vesicle-type studies the reactor was sensitive to wall tissue factor concentration, temperature and the presence of phosphatidylserine in the bilayer.

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