Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody

Rezaie, Alireza R.; Fiore, Martine M.; Neuenschwander, Pierre F.; Esmon, Charles T.; Morrissey, James H.

doi:10.1016/1046-5928(92)90062-2

Cited by 86 publications

(95 citation statements)

References 16 publications

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“…This construction strategy translationally coupled the expression of the neomycin cDNA to that of sTF. To facilitate purification, the 12-residue epitope for the Ca 2ϩ -dependent monoclonal antibody, HPC4, was also linked in-frame to the pelB signal peptide as described previously (6). The Ala substitution mutants of sTF were prepared by PCR mutagenesis methods in the same vector system as described previously (6).…”

Section: Methodsmentioning

confidence: 99%

“…Construction, Mutagenesis, and Expression of Soluble Tissue Factor in Bacteria-Construction and expression of soluble TF lacking both the trans-membrane and cytoplasmic domains (sTF ) in the pINIIIpelB bacterial periplasmic expression/purification vector system has been described previously (6). To improve the expression yield of sTF, this vector was modified by inserting a promoterless neomycin cDNA lacking the Shine-Dalgarno sequence immediately downstream of the sTF 2-219 stop codon.…”

Section: Methodsmentioning

confidence: 99%

“…To facilitate purification, the 12-residue epitope for the Ca 2ϩ -dependent monoclonal antibody, HPC4, was also linked in-frame to the pelB signal peptide as described previously (6). The Ala substitution mutants of sTF were prepared by PCR mutagenesis methods in the same vector system as described previously (6). After confirmation of the accuracy of the mutagenesis by DNA sequencing, the mutant constructs were transformed into the BL21 strain of Escherichia coli and selection was carried out in kanamycin as described previously (6).…”

Section: Methodsmentioning

confidence: 99%

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The Cofactor Function of the N-terminal Domain of Tissue Factor

Kittur

Manithody

Morrissey

et al. 2004

Journal of Biological Chemistry

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Tissue factor (TF) is an integral membrane protein cofactor for factor VIIa (fVIIa) that initiates the blood coagulation cascade during vascular injury. TF has two fibrinonectin type III-like domains, both of which make extensive interactions with both the light and heavy chains of fVIIa. In addition to interaction with fVIIa, the membrane proximal C-terminal domain of TF is also known to bind the natural substrates factors IX and X, thereby facilitating their assembly and recognition by fVIIa in the activation complex. Both fVIIa and TF are elongated proteins, and their complex appears to be positioned nearly perpendicular to the membrane surface. It is possible that, similar to fVIIa, the N-terminal domain of TF also contacts the natural substrates. To investigate this possibility, we substituted all 23 basic and acidic residues of the N-terminal domain of TF with Ala or Asn and expressed the mutants as soluble TF 2-219 in a novel expression/purification vector system in the periplasmic space of bacteria. Following purification to homogeneity, the cofactor properties of mutants in promoting the amidolytic and proteolytic activity of fVIIa were analyzed in appropriate kinetic assays. The amidolytic activity assays indicated that several charged residues spatially clustered at the junction of the N-and C-terminal domains of TF are required for high affinity interaction with fVIIa. On the other hand, the proteolytic activity assays revealed that none of the residues under study may be an interactive site for either factor IX or factor X. However, it was discovered the Arg 74 mutant of TF was defective in enhancing both the amidolytic and proteolytic activity of fVIIa, suggesting that this residue may be required for the allosteric activation of the protease.Tissue factor (TF) 1 is an integral membrane cofactor that upon exposure to circulating blood binds with high affinity to factor VIIa (fVIIa) to catalyze the rapid activation of procoagulant zymogens factors IX and X, thereby initiating the blood clotting cascade (1-3). The structure of TF is composed of two fibrinonectin type III-like extracellular domains, a single membrane-spanning domain and a short cytoplasmic tail (4, 5). Although all three domains of TF are required for the physiological function of the cofactor, previous studies have indicated that the two extracellular domains expressed by recombinant DNA methods as a soluble protein (sTF) can bind to fVIIa with a high affinity to enhance both the amidolytic and proteolytic activities of the protease (6 -9). The crystal structure of sTF either alone or in complex with fVIIa has been determined (4, 10). The structural data have indicated that the extracellular domains of TF make extensive interactions with both the light and heavy chains of fVIIa (10). It is believed that these interactions allosterically change the conformation of the active-site pocket of fVIIa, leading to a dramatic improvement in the catalytic efficiency of the protease toward both synthetic and natural macromolecular substrates (1...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The Cofactor Function of the N-terminal Domain of Tissue Factor

Kittur

Manithody

Morrissey

et al. 2004

Journal of Biological Chemistry

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“…Activation by the Factor VIIa-TF Complex-The initial rate of concentration dependence of FX activation on PC/PS vesicles was studied in both the absence and presence of sTF and dcTF incorporated into PC/PS vesicles as described (35,36). Briefly, the activation of increasing concentrations of the wild-type and mutant FX (7.8 -2600 nM) by factor VIIa (20 nM in the absence of and 0.025-1.0 nM in the presence of TF) was monitored in the absence or presence of a saturating concentration of sTF (250 nM) and relipidated dcTF (2 nM) in TBS/Ca 2ϩ for 1-30 min at room temperature in 30-l reactions in 96-well assay plates.…”

Section: Mutagenesis and Expression Of Recombinant Proteins-constructionmentioning

confidence: 99%

“…After confirmation of the accuracy of the mutagenesis by DNA sequencing, the constructs containing either wild-type or mutant FX cDNA were transfected into HEK293 cells, and the recombinant proteins were isolated from cell culture supernatants by a combination of immunoaffinity and ion exchange chromatography using the HPC4 monoclonal antibody and a Mono Q FPLC column as described (34). Recombinant soluble tissue-factor (sTF) and TF lacking the cytoplasmic domain (dcTF), both purified from bacteria (35,36), were kindly provided by Dr. James Morrissey (University of Illinois at Urbana-Champaign).…”

Section: Mutagenesis and Expression Of Recombinant Proteins-constructionmentioning

confidence: 99%

Role of the N-terminal Epidermal Growth Factor-like Domain of Factor X/Xa

Kittur

Manithody

Rezaie

2004

Journal of Biological Chemistry

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The functional importance of the N-terminal epidermal growth factor-like domain (EGF-N) of factor X/Xa (FX/Xa) was investigated by constructing an FX mutant in which the exon coding for EGF-N was deleted from FX cDNA. Following expression and purification to homogeneity, the mutant was characterized with respect to its ability to function as a zymogen for either the factor VIIa-tissue factor complex or the factor IXa-factor VIIIa complex and then to function as an enzyme in the prothrombinase complex to catalyze the conversion of prothrombin to thrombin. It was discovered that EGF-N is essential for the recognition and efficient activation of FX by both activators in the presence of the cofactors. On the other hand, the FXa mutant interacted with factor Va with a normal apparent dissociation constant and activated prothrombin with ϳ3-fold lower catalytic efficiency in the prothrombinase complex. Surprisingly, the mutant activated prothrombin with ϳ12-fold better catalytic efficiency than wild-type FXa in the absence of factor Va. The mutant was inactive in both prothrombin time and activated partial thromboplastin time assays; however, it exhibited a similar specific activity in a onestage FXa clotting assay. These results suggest that EGF-N of FX is required for the cofactor-dependent zymogen activation by both physiological activators, but it plays no apparent role in FXa recognition of the cofactor in the prothrombinase complex.

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Dual application of cryogel as solid support in peptide synthesis and subsequent protein‐capture

Jespersen¹,

Nielsen

Matthiesen

et al. 2013

J of Applied Polymer Sci

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The use of a cryogel in a combined application as a solid support for automated synthesis of a peptide ligand followed by affinity chromatography of a target protein is evaluated. The advantage, of synthesizing the ligand directly on the cryogel, is the circumvention of the standard process of synthesizing a peptide on a solid support, followed by cleavage, purification, analysis, and finally immobilization on the cryogel. To demonstrate the application, a peptide affinity ligand is synthesized directly on a cryogel with a yield of 28.4 lmol g 21 dry polymer and purity of 45% of crude product. The affinity capture of an antipeptide antibody reveals a specific binding capacity of 0.86 mg g 21 dry polymer. To further elucidate the general availability of a peptide ligand to a macromolecular interaction, a trypsin substrate is synthesized on a cryogel. Trypsin cleavage of immobilized substrate is determined to 1.5 lmol g 21 dry polymer.

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Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody

Cited by 86 publications

References 16 publications

The Cofactor Function of the N-terminal Domain of Tissue Factor

The Cofactor Function of the N-terminal Domain of Tissue Factor

Role of the N-terminal Epidermal Growth Factor-like Domain of Factor X/Xa

Dual application of cryogel as solid support in peptide synthesis and subsequent protein‐capture

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