SUSTAINED EXPRESSION OF FULL LENGTH AND VARIANT RECOMBINANT FACTOR VIII IN GENETICALLY ENGINEERED CELLS

1987 ◽  
Author(s):  
Nava Sarver ◽  
George A Ricca
Keyword(s):  
Factor Viii ◽  
Recombinant Dna ◽  
Expression System ◽  
Genetically Engineered ◽  
Full Length ◽  
Cdna Insert ◽  
Mammalian Expression ◽  
Coagulant Activity ◽  
Recombinant Fviii

A major effort is presently underway to provide factor VIII (FVIII) in a form free of viral pathogens via a recombinant DNA approach. We have constructed two chimeric FVIII cDNA vectors based on the bovine papillomavirus mammalian expression system. The first vector (FVIII) contained a full length FVIII cDNA; the second vector (AFVIII) contained a cDNA insert with an extensive deletion, corresponding to amino acid residues 747 to 1560 in the region encoding the "B" domain. This internal region is removed during activation of the parental FVIII molecule and is believed not to be required for coagulant activity. We have found that recombinant FVIII produced by stable cell lines harboring either the full length or the variant FVIII was capable of restoring coagulant activity to FVIII deficient plasma in. vitro. This expressed activity was neutralized by anti-FVIII antibodies. Similar to observations with FVIII derived from human plasma, the two recombinant FVIII forms were (i) inactivated by the chelating agent EDTA, (ii) demonstrated a biphasic response of an initial activation followed by a decay in activity when treated with thrombin, and (iii) presented the expected peptide banding pattern by western blot analyses. A higher percentage of ΔFVIII transformants were isolated expressing coagulant activity compared to transformants harboring the complete FVIII cDNA. Among the positive transformants isolated, those harboring ΔFVIII produced higher levels of coagulant activity than their full length counterparts. Comparable steady state levels of FVIII specific transcripts were detected in FVIII and ΔFVIII transformants indicating that the difference in expression levels is due to a post transcriptional event(s). Our study demonstrates the efficacy of a full length and an abridged recombinant FVIII produced by stably transformed cells in correcting coagulation deficiency in. vitro. It further suggests the potential usefulness of other molecular variants for efficient expression in genetically engineered cells.

DETERMINATION OF THE DOMAINS OF FACTOR VIII ESSENTIAL FOR PROCOAGULANT ACTIVITY

1987 ◽  
Author(s):  
M Ph Verbeet ◽  
R F Evers ◽  
A Leyte ◽  
H L Lamain ◽  
A J J Van Ooyen ◽  
...  
Keyword(s):  
Factor Viii ◽  
Recombinant Proteins ◽  
Cleavage Site ◽  
Specific Activity ◽  
Expression System ◽  
Full Length ◽  
Procoagulant Activity ◽  
Expression Vectors ◽  
Recombinant Fviii

Factor VIII (FVIII) consists of an obvious domain structure that can be represented as A1-A2-B-A3-C1-C2 (Vehar et al., 1984, Nature 312, 337). In order to determine the domains involved in the procoagulant activity of FVIII, we constructed mutant FVIII cDNAs containing deletions in the coding sequence of the full-length molecule. In one of the mutants a large part of the B domain is deleted. In another one we made a deletion in the B domain that extends beyond the thrombine cleavage site. We used pSV-2 derived expression vectors and COS-1 cells in a transient expression system for the full-length and mutant recombinant proteins. Conditioned media (CM) were harvested.In accordance with the described mutants of recombinant FVIII (Toole et al., 1986, PNAS 83, 5939), we demonstrated an increase in activity in the CM for these mutants as compared to the full-length activity. We also found that the specific activity of the mutants is similar to that of plasma FVIII. So, shorter chains lead to an increased amount of procoagulant protein.

A multi-centre collaborative study on the potency estimation of ReFacto

2003 ◽  
Vol 90 (12) ◽  
pp. 1088-1093 ◽  
Author(s):  
Dawn Sands ◽  
Eva Sandberg ◽  
Rainer Seitz ◽  
Trevor Barrowcliffe ◽  
Anthony Hubbard
Keyword(s):  
Factor Viii ◽  
Collaborative Study ◽  
Full Length ◽  
European Pharmacopoeia ◽  
International Standard ◽  
Assigned Value ◽  
Coagulant Activity ◽  
The Mean ◽  
Good Agreement

SummarySeven laboratories estimated factor VIII coagulant activity in recombinant B-domain-deleted (ReFacto) and plasma-derived FVIII concentrates (Octonativ-M) using chromogenic methods relative to the WHO 6th International Standard FVIII Concentrate (WHO 6th IS), European Pharmacopoeia BRP#2 (EP#2) and the ReFacto Laboratory Standard (RLS). Significantly higher estimates were obtained for all batches of product when calculated relative to the RLS in comparison with estimates vs WHO 6th IS and EP#2. Mean estimates for two batches of ReFacto product vs the RLS were within 10% of the labelled potency whereas estimates vs WHO 6th IS and EP#2 ranged from 21 to 31% lower than the label. Conversely, mean estimates for Octonativ-M relative to WHO 6th IS and EP#2 were within 10% of the label whereas the mean estimate vs RLS was 117% of label. Mean estimates for the ReFacto product, vs the WHO 6th IS and EP#2, varied considerably between the different chromogenic kits whereas estimates vs the RLS showed good agreement between kits. Mean estimates for the RLS vs the WHO 6th IS (8.10 IU/vial) and the EP#2 (7.66 IU/vial) were lower than the assigned value of 9.4 IU/vial. The results are consistent with ReFacto and full-length FVIII responding differently to variations in assay methodology and also indicate that the assigned value on the RLS may be too high. Since this study the unitage on the RLS has been adjusted to effectively increase the amount of ReFacto in the product by 20%.

scholarly journals The Plasmodium falciparum circumsporozoite protein produced in Lactococcus lactis is pure and stable

2019 ◽  
Vol 295 (2) ◽  
pp. 403-414 ◽  
Author(s):  
Susheel K. Singh ◽  
Jordan Plieskatt ◽  
Bishwanath Kumar Chourasia ◽  
Vandana Singh ◽  
Judith M. Bolscher ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Lactococcus Lactis ◽  
Malaria Vaccine ◽  
Vaccine Development ◽  
Expression System ◽  
Surface Protein ◽  
Circumsporozoite Protein ◽  
Full Length

The Plasmodium falciparum circumsporozoite protein (PfCSP) is a sporozoite surface protein whose role in sporozoite motility and cell invasion has made it the leading candidate for a pre-erythrocytic malaria vaccine. However, production of high yields of soluble recombinant PfCSP, including its extensive NANP and NVDP repeats, has proven problematic. Here, we report on the development and characterization of a secreted, soluble, and stable full-length PfCSP (containing 4 NVDP and 38 NANP repeats) produced in the Lactococcus lactis expression system. The recombinant full-length PfCSP, denoted PfCSP4/38, was produced initially with a histidine tag and purified by a simple two-step procedure. Importantly, the recombinant PfCSP4/38 retained a conformational epitope for antibodies as confirmed by both in vivo and in vitro characterizations. We characterized this complex protein by HPLC, light scattering, MS analysis, differential scanning fluorimetry, CD, SDS-PAGE, and immunoblotting with conformation-dependent and -independent mAbs, which confirmed it to be both pure and soluble. Moreover, we found that the recombinant protein is stable at both frozen and elevated-temperature storage conditions. When we used L. lactis–derived PfCSP4/38 to immunize mice, it elicited high levels of functional antibodies that had the capacity to modify sporozoite motility in vitro. We concluded that the reported yield, purity, results of biophysical analyses, and stability of PfCSP4/38 warrant further consideration of using the L. lactis system for the production of circumsporozoite proteins for preclinical and clinical applications in malaria vaccine development.

Alternatively spliced human tissue factor (asHTF) is not pro-coagulant

2007 ◽  
Vol 97 (01) ◽  
pp. 11-14 ◽  
Author(s):  
Petra Censarek ◽  
Anett Bobbe ◽  
Maria Grandoch ◽  
Karsten Schrör ◽  
Artur-Aron Weber
Keyword(s):  
Smooth Muscle ◽  
Tissue Factor ◽  
Human Tissue ◽  
Thrombin Generation ◽  
Expression System ◽  
Hek293 Cells ◽  
Transfected Cells ◽  
Mammalian Expression ◽  
Coagulant Activity ◽  
Alternatively Spliced

SummaryIt has been proposed that alternatively-spliced human tissue factor (asHTF) is pro-coagulant. We have evaluated the function of asHTF in a mammalian expression system. Full-length human tissue factor (HTF) and asHTF were cloned from smooth muscle cells and over-expressed in HEK293 cells. As expected, a marked pro-coagulant activity (FX activation, thrombin generation) was observed on the surface, in lysates, and on microparticles from HTF transfected cells. In contrast, no pro-coagulant activity of as HTF was observed.

ADAMTS13 Binds VWF Via its C-Terminal CUB2 Domain.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 126-126 ◽  
Author(s):  
Weirui Zhang ◽  
David Motto ◽  
David Ginsburg
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Expression Vector ◽  
Full Length ◽  
Mammalian Expression ◽  
Partial Denaturation

Abstract Thrombotic thrombocytopenic purpura (TTP) is a life threatening illness due to a deficiency of the VWF-cleaving protease, ADAMTS13. The ADAMTS13 protein is composed of a propeptide, followed by a typical zinc metalloprotease domain. The C-terminal 2/3 of the molecule contains disintegrin-like, cystine-rich, and spacer domains, as well as a total of eight TSP1 motifs and two CUB domains. The function of this C-terminal portion of the molecule and its composite motifs is unknown, though TSP1 and CUB domains of other proteins have been shown to mediate protein-protein interactions. To further explore the interaction between ADAMTS13 and VWF, we cloned full length human cDNAs for both ADAMTS13 and VWF into the mammalian expression vector pcDNA3.1. These constructs were transiently transfected into 293T cells and COS cells respectively, and conditioned media collected for analysis. Using an anti-myc antibody, myc-tagged VWF co-immunoprecipitated (co-IP) with ADAMTS13, as demonstrated by western blot analysis using antisera raised against a C-terminal peptide derived from the predicted ADAMTS13 sequence. This direct interaction required partial denaturation of VWF in 1M urea, with no co-IP observed in the absence of urea. To map the segment within ADAMTS13 responsible for VWF binding, we cloned a series of overlapping ADAMTS13 fragments into the bacterial expression vector, Pet44b. Fusion proteins were purified by binding of the included His-tag to Ni-NTA beads and incubated with recombinant myc-VWF in the presence of 1M urea. Association with VWF was analyzed by co-IP with anti-myc followed by western blot analysis using an antibody to the C-terminal HSV-tag present in each fusion protein. The CUB2 (Glu1298- Thr1427) fusion protein co-IP’d with full-length VWF and also demonstrated concentration-dependent competition with full-length ADAMTS13 for VWF binding. In summary, we have demonstrated a direct protein-protein interaction between VWF and ADAMTS13. Binding requires partial denaturation of VWF and appears to be mediated primarily through contacts with the ADAMTS13 CUB2 domain. This interaction may account for the previously observed co-purification of VWF and ADAMTS13 from human plasma. Furthermore, the requirement for 1M urea suggests that this interaction may only occur physiologically under conditions of high shear. Though others have shown that the C-terminal domains of ADAMTS13, including CUB2, are not required for VWF cleavage in vitro, our data, together with several C-terminal mutations previously reported in TTP patients, suggest that interactions between VWF and the ADAMTS13 CUB2 domain may be important in vivo.

Confocal microscopy analysis of native, full length and B-domain deleted coagulation factor VIII trafficking in mammalian cells

2004 ◽  
Vol 92 (07) ◽  
pp. 23-35 ◽  
Author(s):  
Sven Becker ◽  
Jeremy Simpson ◽  
Rainer Pepperkok ◽  
Stefan Heinz ◽  
Christian Herder ◽  
...  
Keyword(s):  
Factor Viii ◽  
Golgi Complex ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Coagulation Factor ◽  
Full Length ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Analysis ◽  
Recombinant Fviii

SummaryIn mammalian cells, factor VIII (FVIII) secretion depends upon its interaction with chaperones of the endoplasmic reticulum (ER) and requires a unique ATP-dependent step to dissociate aggregates formed within the ER. To further elucidate mechanisms which might account for the inefficient secretion of recombinant FVIII (rFVIII), we have analyzed the pathways of recombinant full length (rFVIII-FL) and B-domain deleted (rFVIIIΔB) FVIII and compared these to the secretion route of native FVIII in primary hepatocytes. Using confocal laser scanning microscopy in combination with a pulse chase of a known secretion marker, we describe the trafficking route of FVIII, which upon release from the ER – where it colocalizes with calnexin – is transported to the Golgi complex in vesiculartubular transport complexes (VTCs) which could be further identified as being COP I coated. However, a large portion of rFVIII is retained in the ER and additionally in structures which could not be assigned to the ER, Golgi complex or intermediate compartment. Moderate BiP transcription levels indicate that this observed retention of FVIII does not reflect cellular stress due to an overexpression of FVIII-protein in transduced cells. Moreover, a pulse of newly synthesized rFVIII protein is released within 4 hrs, indicating that once rFVIII is released from the ER there is no further limitation to its secretion. Our data provide new details about the secretory route of FVIII, which may ultimately help to identify factors currently limiting the efficient and physiological expression of FVIII in gene therapy and manufacture.

scholarly journals Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates

2021 ◽  
Vol 17 (7) ◽  
pp. e1008864
Author(s):  
Duncan N. Ndegwa ◽  
Prasun Kundu ◽  
Jessica B. Hostetler ◽  
Alejandro Marin-Menendez ◽  
Theo Sanderson ◽  
...  
Keyword(s):  
Plasmodium Vivax ◽  
Culture System ◽  
Vaccine Development ◽  
Expression System ◽  
Critical Role ◽  
Blood Stage ◽  
Effective Vaccine ◽  
Mammalian Expression ◽  
Close Phylogenetic Relationship

Plasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up.

Validation of a Procedure for Potency Assessing of a High Purity Factor VIII Concentrate -Comparison of Different Factor VIII Coagulant Assays and Effect of Prediluent

1990 ◽  
Vol 64 (02) ◽  
pp. 251-255 ◽  
Author(s):  
Claudine Mazurier ◽  
Armelle Parquet-Gernez ◽  
Maurice Goudemand
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Assay Method ◽  
One Stage ◽  
Chromogenic Assay ◽  
Coagulant Activity ◽  
The One ◽  
In Vitro Data

SummaryThe assessment of factor VIII coagulant activity (FVTII: C) in recently available highly purified and concentrated FVTII therapeutic products calls for careful evaluation of assay methodologies. We assayed more than 130 batches of a concentrate with a specific activity of about 150 FVTII :C units/mg protein, using one-stage and two-stage clotting and chromogenic methods. There was good agreement between the potency estimates obtained with the different methods. We also compared the FVTII :C potencies obtained after predilution in buffer or FVIII-deficient plasma using either calibrated plasma or FVTII concentrate as references. With the one-stage assay we found a marked discrepancy between the potency values obtained with buffer and with FVTII-deficient plasma used as prediluents. In order to validate our “in vitro” data we performed 6 “in vivo” analyses in severe haemophilia A patients. On the basis of the overall data obtained we chose to label FVIII potency by using FVIII-deficient plasma as prediluent, reference plasma as standard and the chromogenic assay method.

Pharmacokinetic Properties of Recombinant Factor VIII Compared with a Monoclonally Purified Concentrate (Hemofil® M)

1992 ◽  
Vol 68 (04) ◽  
pp. 433-435 ◽  
Author(s):  
M Morfini ◽  
G Longo ◽  
A Messori ◽  
M Lee ◽  
G White ◽  
...  
Keyword(s):  
Factor Viii ◽  
Half Life ◽  
Hemophilia A ◽  
Recombinant Dna ◽  
Volume Of Distribution ◽  
Pharmacokinetic Properties ◽  
Recombinant Fviii ◽  
Reported Data ◽  
In Vivo Recovery

SummaryA recombinant FVIII preparation, Recombinate™, was compared with a high-purity plasma-derived concentrate, Hemofil® M, in 47 hemophilia A patients in a cross-over evaluation of pharmacokinetic properties. The recombinant material showed a significantly lower clearance, volume of distribution, and higher in vivo recovery, but a similar half-life to the plasma-based product.In a comparison with reported data from other standard concentrates, the recombinant preparation exhibited potentially better pharmacokinetic properties in that its clearance was slower and its half-life was longer.We conclude that the recombinant DNA method of preparation does not adversely affect the biological and pharmacological characteristics of the factor VIII molecule.

Evaluation of an Adenoviral Vector Encoding Full-Length Human Factor VIII in Hemophiliac Mice

1999 ◽  
Vol 81 (02) ◽  
pp. 234-239 ◽  
Author(s):  
Sheila Connelly ◽  
Julie Andrews ◽  
Angela Gallo-Penn ◽  
Luigina Tagliavacca ◽  
Randal Kaufman ◽  
...  
Keyword(s):  
Factor Viii ◽  
Adenoviral Vector ◽  
Human Factor ◽  
Hemophilia A ◽  
Adenoviral Vectors ◽  
Full Length ◽  
Human Factor Viii ◽  
Immunohistochemical Analyses

SummaryAdenoviral vectors provide a promising gene therapy system for the treatment of hemophilia A. Potent vectors encoding a human factor VIII (FVIII) cDNA were developed that mediated sustained FVIII expression in normal and hemophiliac mice and complete phenotypic correction of the bleeding disorder in hemophiliac mice and dogs (Connelly and Kaleko, Haemophilia 1998; 4: 380-8). However, these studies utilized vectors encoding a truncated version of the human FVIII cDNA lacking the B-domain (BDD FVIII). In this work, an adenoviral vector encoding the human full-length (FL) FVIII cDNA was generated and characterized. While functional FL FVIII was secreted in vitro, expression of the FL protein was not detected in the plasma of vector-treated hemophiliac mice. Unexpectedly, the FL FVIII vector-treated animals demonstrated phenotypic correction of the bleeding defect as measured by a tail-clip survival study. FL FVIII protein was visualized in the mouse livers using human FVIII-specific immunohistochemical analyses. These data demonstrate that adenoviral vector-mediated in vivo expression of BDD FVIII is more efficient than that of the FL protein and that phenotypic correction can occur in the absence of detectable levels of FVIII.

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