Expression, purification and characterization of factor IX derivatives using a novel vector system

Yang, Likui; Gopalakrishna, Kota N.; Manithody, Chandrashekhara; Rezaie, Alireza R.

doi:10.1016/j.pep.2006.05.012

Cited by 9 publications

(39 citation statements)

References 36 publications

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“…Proteins-The construction, expression, and purification of wild-type FIX in a novel expression/purification vector system in which the P1Ј-P3Ј residues of the first factor XIa cleavage site on the activation peptide of FIX (Ala 146 -Glu-Ala) have been replaced with the corresponding basic residues of FX (Arg-LysArg) has been described previously (30). This strategy introduces a post-translational processing site for the furin-like enzymes on the activation peptide of FIX, thus the mutant is synthesized as a two-chain zymogen held together by a disulfide bond (30).…”

Section: Construction Mutagenesis and Expression Of Recombinantmentioning

confidence: 99%

Section: Construction Mutagenesis and Expression Of Recombinantmentioning

confidence: 99%

“…This strategy introduces a post-translational processing site for the furin-like enzymes on the activation peptide of FIX, thus the mutant is synthesized as a two-chain zymogen held together by a disulfide bond (30). In this construct, the first 12 residues of the activation peptide of FIX, immediately following the engineered P3Ј Arg, have been replaced with the 12-residue epitope for the Ca 2ϩ -dependent monoclonal antibody, HPC4, in order to facilitate the purification of recombinant FIX by this antibody as described before (30). An FIX mutant in which the residues of the 39-loop from residues Val 31 to Phe 41 (Val 31 -Val-Leu-AsnGly-Lys-Val-Asp-Ala-Phe 41 ) in the chymotrypsin numbering (29) were replaced with the same 39-loop residues of FXa (Ala 31 -Leu-Leu-Ilu-Asn-Glu-Glu-Asn-Glu-Gly-Phe 41 ) (FIXfX 39-loop ) was prepared and expressed in HEK-293 cells using the same expression/purification vector system.…”

Section: Construction Mutagenesis and Expression Of Recombinantmentioning

confidence: 99%

“…An FIX mutant in which the residues of the 39-loop from residues Val 31 to Phe 41 (Val 31 -Val-Leu-AsnGly-Lys-Val-Asp-Ala-Phe 41 ) in the chymotrypsin numbering (29) were replaced with the same 39-loop residues of FXa (Ala 31 -Leu-Leu-Ilu-Asn-Glu-Glu-Asn-Glu-Gly-Phe 41 ) (FIXfX 39-loop ) was prepared and expressed in HEK-293 cells using the same expression/purification vector system. The FIX derivatives were activated by the FX-activating enzyme from Russell's viper venom (RVV-X), and the active enzymes were purified to homogeneity and active-site titrated using a calibrated concentration of AT as described before (30). The Ser 195 to Ala substitution mutant of FIX was constructed using the same vector, activated by RVV-X and purified on SBTI-Sepharose column as described (31).…”

Section: Construction Mutagenesis and Expression Of Recombinantmentioning

confidence: 99%

“…Cleavage of the Chromogenic Substrate-The steady-state kinetics of hydrolysis of CBS 31.39 (0.1-10 mM) by FIXa derivatives (10 nM) was monitored in 0.02 M Tris-HCl (pH 7.5) buffer containing 0.1 M NaCl, 0.1 mg/ml bovine serum albumin (BSA), 33% ethylene glycol, and 5 mM Ca 2ϩ as described (30). The rate of hydrolysis was measured at 405 nm at room temperature in 96-well plates by a V max Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA) as described (30).…”

Section: Construction Mutagenesis and Expression Of Recombinantmentioning

confidence: 99%

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Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

Yang

Manithody

Qureshi

et al. 2010

Journal of Biological Chemistry

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The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300-to 500-fold. Relative to FXa, AT inhibits FIXa with ϳ40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31-41) may partly be responsible for the differential reactivity of the two proteases with AT. This loop is highly acidic in FXa, containing three Glu residues at positions 36, 37, and 39. By contrast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. To determine whether differences in the residues of this loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease was replaced with the corresponding loop of FXa. The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalytic efficiency, however, the mutant exhibited ϳ5-fold enhanced reactivity with AT specifically in the absence of the cofactor, heparin. Further studies revealed that the FIXa mutant activates factor X with ϳ4-fold decreased k cat and ϳ2-fold decreased K m , although the mutant interacted normally with factor VIIIa. Based on these results we conclude that residues of the 39-loop regulate the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate factor X.Factor IXa (FIXa) 2 is a vitamin K-dependent plasma serine protease that, upon complex formation with factor VIIIa (FVIIIa), on negatively charged membrane surfaces in the presence of Ca 2ϩ (intrinsic Tenase) activates factor X (FX) to factor Xa (FXa) during the blood coagulation process (1-5). FIXa plays an important role in the clotting cascade, because its deficiency is associated with the life-threatening disease, hemophilia B (6). The activity of FIXa toward its physiological substrate FX is very poor in the absence of FVIIIa, however, complex formation with the cofactor improves the catalytic efficiency of FIXa by 4 -5 orders of magnitude in the intrinsic Tenase complex (1-5). The proteolytic activity of FIXa in plasma is regulated by the serpin inhibitor, antithrombin (AT) (7)(8)(9)(10)(11). Surprisingly, in contrast to a dramatic cofactor-mediated improvement in the activity of FIXa toward FX, FVIIIa has a minimal cofactor effect on the reactivity of the protease with AT or its activity toward small synthetic substrates (12, 13), suggesting that FVIIIa-mediated exosite interactions with FX, involving sites remote from the active-site pocket, play dominant roles in the catalytic reaction (14, 15). In the case of the FIXa reaction with AT, the cofactor function of heparin-like glycosaminoglycans, similar to those lining the surface of the endothelium (16), is required to facilitate the protease recognition of the serpin (17-19).Heparin accelerates the reactivity of AT with FIXa by two distinct mechanisms: a conformational ac...

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Section: Construction Mutagenesis and Expression Of Recombinantmentioning

confidence: 99%

Section: Construction Mutagenesis and Expression Of Recombinantmentioning

confidence: 99%

Section: Construction Mutagenesis and Expression Of Recombinantmentioning

confidence: 99%

Section: Construction Mutagenesis and Expression Of Recombinantmentioning

confidence: 99%

Section: Construction Mutagenesis and Expression Of Recombinantmentioning

confidence: 99%

See 3 more Smart Citations

Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

Yang

Manithody

Qureshi

et al. 2010

Journal of Biological Chemistry

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Stable and high‐level production of recombinant Factor IX in human hepatic cell line

Fernandes

Fontes

Gonsales

et al. 2011

Biotech and App Biochem

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Hemophilia B is a genetic disease of the coagulation system that affects one in 30,000 males worldwide. Recombinant human Factor IX (rhFIX) has been used for hemophilia B treatment, but the amount of active protein generated by these systems is inefficient, resulting in a high-cost production of rhFIX. In this study, we developed an alternative for rhFIX production. We used a retrovirus system to obtain two recombinant cell lines. We first tested rhFIX production in the human embryonic kidney 293 cells (293). Next, we tested a hepatic cell line (HepG2) because FIX is primarily expressed in the liver. Our results reveal that intracellular rhFIX expression was more efficient in HepG2/rhFIX (46%) than in 293/rhFIX (21%). The activated partial thromboplastin time test showed that HepG2/rhFIX expressed biologically active rhFIX 1.5 times higher than 293/rhFIX (P = 0.016). Recovery of rhFIX from the HepG2 by reversed-phase chromatography was straightforward. We found that rhFIX has a pharmacokinetic profile similar to that of FIX purified from human plasma when tested in hemophilic B model. HepG2/rhFIX cell line produced the highest levels of rhFIX, representing an efficient in vitro expression system. This work opens up the possibility of significantly reducing the costs of rhFIX production, with implications for expanding hemophilia B treatment in developing countries.

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Industrial production of clotting factors: Challenges of expression, and choice of host cells

Kumar

2015

Biotechnology Journal

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The development of recombinant forms of blood coagulation factors as safer alternatives to plasma derived factors marked a major advance in the treatment of common coagulation disorders. These are complex proteins, mostly enzymes or co-enzymes, involving multiple post-translational modifications, and therefore are difficult to express. This article reviews the nature of the expression challenges for the industrial production of these factors, vis-à-vis the translational and post-translational bottlenecks, as well as the choice of host cell lines for high-fidelity production. For achieving high productivities of vitamin K dependent proteins, which include factors II (prothrombin), VII, IX and X, and protein C, host cell limitation of γ-glutamyl carboxylation is a major bottleneck. Despite progress in addressing this, involvement of yet unidentified protein(s) impedes a complete cell engineering solution. Human factor VIII expresses at very low levels due to limitations at several steps in the protein secretion pathway. Protein and cell engineering, vector improvement and alternate host cells promise improvement in the productivity. Production of Von Willebrand factor is constrained by its large size, complex structure, and the need for extensive glycosylation and disulfide-bonded oligomerization. All the licensed therapeutic factors are produced in CHO, BHK or HEK293 cells. While HEK293 is a recent adoption, BHK cells appear to be disfavored.

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Expression, purification and characterization of factor IX derivatives using a novel vector system

Cited by 9 publications

References 36 publications

Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

Stable and high‐level production of recombinant Factor IX in human hepatic cell line

Industrial production of clotting factors: Challenges of expression, and choice of host cells

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