Prolonged in vivo anticoagulant activity of a hirudin–albumin fusion protein secreted from Pichia pastoris

Sheffield, William P.; Smith, Ian; Syed, Summer; Bhakta, Varsha

doi:10.1097/00001721-200109000-00003

Cited by 31 publications

(35 citation statements)

References 33 publications

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“…Purified plasma-derived, enzymatically treated, or recombinant forms of AGP were iodinated using the Iodogen method [31] and injected into the marginal ear vein of rabbits, as previously described [32]. At timed intervals (0.083, 0.5, 1, 2, 4, 6, 8, 24, 48, 72, 96, and 168 hours or until the recovered radioactivity declined lower than 0.1% of the injected dose) blood samples were taken from the marginal vein of the other ear, centrifuged to obtain plasma, and the trichloroacetic acid-precipitable radioactivity was determined by γ counting.…”

Section: Methodsmentioning

confidence: 99%

In VivoClearance of Alpha-1 Acid Glycoprotein Is Influenced by the Extent of Its N-Linked Glycosylation and by Its Interaction with the Vessel Wall

McCurdy

Bhakta

Eltringham-Smith

et al. 2012

Journal of Biomedicine and Biotechnology

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Alpha-1 acid glycoprotein (AGP) is a highly glycosylated plasma protein that exerts vasoprotective effects. We hypothesized that AGP’s N-linked glycans govern its rate of clearance from the circulation, and followed the disappearance of different forms of radiolabeled human AGP from the plasma of rabbits and mice. Enzymatic deglycosylation of human plasma-derived AGP (pdAGP) by Peptide: N-Glycosidase F yielded a mixture of differentially deglycosylated forms (PNGase-AGP), while the introduction of five Asn to Gln mutations in recombinantPichia pastoris-derived AGP (rAGP-N(5)Q) eliminated N-linked glycosylation. PNGase-AGP was cleared from the rabbit circulation 9-fold, and rAGP-N(5)Q, 46-fold more rapidly than pdAGP, primarily via a renal route.Pichia pastoris-derived wild-type rAGP differed from pdAGP in expressing mannose-terminated glycans, and, like neuraminidase-treated pdAGP, was more rapidly removed from the rabbit circulation than rAGP-N(5)Q. Systemic hyaluronidase treatment of mice transiently decreased pdAGP clearance. AGP administration to mice reduced vascular binding of hyaluronic acid binding protein in the liver microcirculation and increased its plasma levels. Our results support a critical role of N-linked glycosylation of AGP in regulating itsin vivoclearance and an influence of a hyaluronidase-sensitive component of the vessel wall on its transendothelial passage.

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Section: Methodsmentioning

confidence: 99%

In VivoClearance of Alpha-1 Acid Glycoprotein Is Influenced by the Extent of Its N-Linked Glycosylation and by Its Interaction with the Vessel Wall

McCurdy

Bhakta

Eltringham-Smith

et al. 2012

Journal of Biomedicine and Biotechnology

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“…The 360 bp amplification product was restricted with XhoI and EcoRI and the 204 bp restriction product was restricted with, and inserted between, these sites in pPICZ9ssamp [26] to yield pPIC9zssKPIH 6 . This plasmid was used exactly as described above for pPICZ9ssKPIHSAH 6 in directing the expression and purification of recombinant KPI (63 amino acids), comprising the 57 amino acids of KPI and a C-terminal hexahistidine tag.…”

Section: Methodsmentioning

confidence: 99%

Fusion to Human Serum Albumin Extends the Circulatory Half-Life and Duration of Antithrombotic Action of the Kunitz Protease Inhibitor Domain of Protease Nexin 2

Sheffield¹,

Eltringham-Smith²,

Bhakta³

2018

Cell Physiol Biochem

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Background/Aims: The Kunitz Protease Inhibitor (KPI) domain of protease nexin 2 (PN2) potently inhibits coagulation factor XIa. Recombinant KPI has been shown to inhibit thrombosis in mouse models, but its clearance from the murine circulation remains uncharacterized. The present study explored the pharmacokinetic and pharmacodynamic effects of fusing KPI to human serum albumin (HSA) in fusion protein KPIHSA. Methods: Hexahistidine-tagged KPI (63 amino acids) and KPIHSA (656 amino acids) were expressed in Pichia pastoris yeast and purified by nickel-chelate chromatography. Clearance profiles in mice were determined, as well as the effects of KPI or KPIHSA administration on FeCl₃-induced vena cava thrombus size or carotid artery time to occlusion, respectively. Results: Fusion to HSA increased the mean terminal half-life of KPI by 8-fold and eliminated its interaction with the low density lipoprotein receptor-related protein. KPI and KPIHSA similarly reduced thrombus size and occlusion in both venous and arterial thrombosis models when administered at the time of injury, but only KPI was effective when administered one hour before injury. Conclusions: Albumin fusion deflects KPI from rapid in vivo clearance without impairing its antithrombotic properties and widens its potential therapeutic window.

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“…HSA fusion proteins were purified from media conditioned by transformed Pichia pastoris cells and induced with 0.5% vol/vol methanol, as previously described [18,19,28], except that the methanol induction phase of protein production was extended to 96 hours. At that point, the conditioned media was neutralized and clarified by centrifugation [1], treated with protease inhibitors (5 mM benzamidine and 0.1 mM phenylmethylsulfonyl fluoride), and purified using Ni-NTA agarose chromatography (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa

Sheffield

Eltringham-Smith

2011

BMC Biotechnol

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BackgroundThe transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α2-antiplasmin (α2AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α2AP residues 13-42 linked to human serum albumin (HSA) weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α2AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa.ResultsExpression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H6NQEQVSPLTLLAG4Y (designated XL1); H6DQMMLPWAVTLG4Y (XL2); H6WQHKIDLPYNGAG4Y (XL3); and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6). The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α2AP(13-42)-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA) most rapidly by FXIIIa, and was the most effective competitor of α2AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA.ConclusionsFusion protein XL5-HSA (DQMMLPWAVTLG4Y-HSAH6) was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in vitro. The improved stability and reactivity of this chimeric protein was further evidenced by its incorporation into in vivo clots formed in thrombosis models in both mice and rabbits.

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Prolonged in vivo anticoagulant activity of a hirudin–albumin fusion protein secreted from Pichia pastoris

Cited by 31 publications

References 33 publications

In VivoClearance of Alpha-1 Acid Glycoprotein Is Influenced by the Extent of Its N-Linked Glycosylation and by Its Interaction with the Vessel Wall

In VivoClearance of Alpha-1 Acid Glycoprotein Is Influenced by the Extent of Its N-Linked Glycosylation and by Its Interaction with the Vessel Wall

Fusion to Human Serum Albumin Extends the Circulatory Half-Life and Duration of Antithrombotic Action of the Kunitz Protease Inhibitor Domain of Protease Nexin 2

Incorporation of albumin fusion proteins into fibrin clots in vitro and in vivo: comparison of different fusion motifs recognized by factor XIIIa

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