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Biochemical and Biomolecular Engineering Commons™
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Articles 31 - 39 of 39
Full-Text Articles in Biochemical and Biomolecular Engineering
Affinity Purification Of Biologically Active Andinactive Forms Of Recombinant Human Protein C Produced In Porcine Mammary Gland, Kevin E. Van Cott, Barry Williams Department Of Chemical Engineering,Virginia Tech University, Blacksburg, Va 24061, Usa, William H. Velander, Francis C. Gwazdauskas, Tim Lee Jerome Holland Laboratory, American Red Cross, Rockville, Md 20855, Usa, Henryk Lubon, William N. Drohan
Affinity Purification Of Biologically Active Andinactive Forms Of Recombinant Human Protein C Produced In Porcine Mammary Gland, Kevin E. Van Cott, Barry Williams Department Of Chemical Engineering,Virginia Tech University, Blacksburg, Va 24061, Usa, William H. Velander, Francis C. Gwazdauskas, Tim Lee Jerome Holland Laboratory, American Red Cross, Rockville, Md 20855, Usa, Henryk Lubon, William N. Drohan
Papers in Biotechnology
Recombinant human protein C (rhPC) secreted in the milk of transgenic pigs was studied. 'Ikansgenes having different regulatory elements of the murine milk protein, whey acidic protein, were used with cDNA and genomic human protein C (hPC) DNA sequences to obtain lower and higher expressing animals. The cDNA pigs had a range of expression of about 0.1-0.5 g/l milk. Two different genomic hPC pig lines have expressed 0.3 and 1-2 g/l, respectively. The rhPC was first purified at yields greater than 60 per cent using a monoclonal antibody (mAb) to the activation site on the heavy chain of hPC. Subsequent …
Effect Of Antibody Orientation On Immunosorbent, Anuradha Subramanian, William H. Velander
Effect Of Antibody Orientation On Immunosorbent, Anuradha Subramanian, William H. Velander
Papers in Biotechnology
The impact of antibody orientation on immunosorbent efficiency was quantitatively assessed. A pH-dependent murine monoclonal antibody (Mab) against human protein C (hPC), recombinant hPC (rhPC) and two different immobilization chemistries and matrices were used as model systems. The lysyl groups of the rhPC were covalently modified with an acetic acid ester of N-hydroxysuccinimide and this modified rhPC was used as a Fab masking agent (FMA). The FMA was used to mask the antigen bind-ing regions (Fab) of the Mab prior to and during covalent immobilization. Thereafter, the residual active sites of the support were in-activated and the FMA was removed. …
Expression Of A Functional Human Complement Inhibitor In A Transgenic Pig As A Model For The Prevention Of Xenogeneic Hyperacute Organ Rejection., William L. Fodor, Barry L. Williams, Louis A. Martis, Josaeph A. Madri, Scott A. Rollins, James W. Knight, William H. Velander, Stephen P. Squinto
Expression Of A Functional Human Complement Inhibitor In A Transgenic Pig As A Model For The Prevention Of Xenogeneic Hyperacute Organ Rejection., William L. Fodor, Barry L. Williams, Louis A. Martis, Josaeph A. Madri, Scott A. Rollins, James W. Knight, William H. Velander, Stephen P. Squinto
Papers in Biotechnology
ABSTRACT The serious shortage of human organs available for transplantation has engendered a heightened interest in the use of animal organs (xenografts) for transplantation. However, the major barrier to successful discordant xenogeneic organ transplantation is the phenomenon of hyperacute rejection. Hyperacute rejection results from the deposition of high-titer preformed antibodies that activate serum complement on the luminal surface of the vascular endothelium, leading to vessel occlusion and graft failure within minutes to hours. Although endogenous membrane-associated complement inhibitors normally protect endothelial cells from autologous complement, they are species restricted and thus confer limited resistance to activated xenogeneic complement. To address …
Effect Of Culture Conditions, Donor Age, And Injection Site On In Vitro Development Of Dna Microinjected Porcine Zygotes, M A. Hajdu, J W. Knight, R S. Cansecot, R L. Krisher, William H. Velander, R E. Pearson, F C. Gwazdauskas
Effect Of Culture Conditions, Donor Age, And Injection Site On In Vitro Development Of Dna Microinjected Porcine Zygotes, M A. Hajdu, J W. Knight, R S. Cansecot, R L. Krisher, William H. Velander, R E. Pearson, F C. Gwazdauskas
Papers in Biotechnology
A series of experiments evaluated development of porcine zygotes microinjected with DNA in three culture media and two incubation temperatures, from postpubertal and prepubertal donors, and between zygotes injected with DNA into the pronucleus and the cytoplasm. Zygotes recovered from 36 postpubertal gilts in Exp. 1 were injected and cultured in modified NCSU-23, modified NCSU-37, and CZB media at 37°C or 39°C for 7 d. In Exp. 2, zygotes were collected from postpubertal or prepubertal gilts, microinjected with DNA, and cultured in modified NCSU-23. In Exp. 3 superovulated prepubertal gilts had DNA injected into the cytoplasm or pronucleus of zygotes. …
Effects Of Time Of Deoxyribonucleic Acid Microinjection On Gene Detection And In Vitro Development Of Bovine Embryos, A.E T. Sparks, R S. Canseco, C G. Russell, J L. Johnson, H D. Moll, William H. Velander, Francis C. Gwazdauskas
Effects Of Time Of Deoxyribonucleic Acid Microinjection On Gene Detection And In Vitro Development Of Bovine Embryos, A.E T. Sparks, R S. Canseco, C G. Russell, J L. Johnson, H D. Moll, William H. Velander, Francis C. Gwazdauskas
Papers in Biotechnology
In vivo fertilized embryos were surgically collected from superovulated dairy cows to evaluate microinjection on embryo development and utilized the polymerase chain reaction technique for selection of transgenic embryos. Seventy-two percent of the embryos with visible pronuclei or nuclei were microinjected with DNA, and the remaining 28% served as uninjected controls. All embryos were cocultured with bovine oviductal epithelial cells. Mean final development scores of embryos within the same initial cell stage at collection were unaffected by microinjection. After 144 h of culture, 45% of the microinjected embryos developed to the morula or blastocyst stage. The transgene was detected in …
High-Level Expression Of A Heterologous Protein In The Milk Of Transgenic Swine Using The Cdna Encoding Human Protein C, William H. Velander, John L. Johnson, Raymond L. Page, Chritopher G. Russell, Anuradha Subramanian, Tracy D. Wilkins, Francis C. Gwazdauskas, Christoph Pittus, William N. Drohan
High-Level Expression Of A Heterologous Protein In The Milk Of Transgenic Swine Using The Cdna Encoding Human Protein C, William H. Velander, John L. Johnson, Raymond L. Page, Chritopher G. Russell, Anuradha Subramanian, Tracy D. Wilkins, Francis C. Gwazdauskas, Christoph Pittus, William N. Drohan
Papers in Biotechnology
Transgenic pigs were generated that produced human protein C in their milk at up to 1 g/liter. The gene construct was a fusion gene consisting of the cDNA for human protein C inserted into the first exon of the mouse whey acidic protein gene. These results demonstrate that the mouse whey acidic protein gene contains regulatory elements that can direct cDNA expression at high levels in the pig mammary gland. Recombinant human protein C that was produced at about 380 pg/ml per hr in transgenic pig milk possessed anticoagulant activity that was equivalent to that of protein C derived from …
In Vitro Development Of Zygotes From Prepubertal Gilts After Microinjection Of Dna, B. L. Williams, J. L. Johnson, A. E.T. Sparks, R. S. Canseco, J. W. Knight, William H. Velander, R. L. Page, W. N. Drohan, J. M. Young, R. E. Pearson, T. D. Wilkins, F. C. Gwazdauskas
In Vitro Development Of Zygotes From Prepubertal Gilts After Microinjection Of Dna, B. L. Williams, J. L. Johnson, A. E.T. Sparks, R. S. Canseco, J. W. Knight, William H. Velander, R. L. Page, W. N. Drohan, J. M. Young, R. E. Pearson, T. D. Wilkins, F. C. Gwazdauskas
Papers in Biotechnology
The effect of pronuclear microinjection of DNA and culture in excised mouse oviducts on the development of porcine zygotes was assessed in this study. Precocious ovulation was induced in prepubertal gilts with pregnant mare's serum gonadotrophin and hCG. Zygotes received either pronuclear microinjection of buffer alone, buffer containing a DNA construct, or no microinjection. Zygotes were cultured in vitro in either modified Krebs-Ringer bicarbonate medium (KRBI for 144 h or in mouse oviduct (MO) explant culture with KRB for 48, 72, 96, or 120 h. Pronuclear microinjection of DNA resulted in a lower (P < .05) cleavage index (CII than did buffer or no microinjection (CI 2.16 k .10 vs 2.80 f .13 and 2.93 f .lo). The CI loss was greatest for DNA-injected zygotes at the two-cell stage of development. Coculture of zygotes in MO resulted in a higher CI (P < .01) than did culture in KRB. Culture in MO for 72 h was the most beneficial system compared with MO for 48, 96, or 120 h (P < .05; CI 3.25 k .12 vs 2.66 k .18, 2.79 f .14, and 2.40 .14, respectively). Microinjection of DNA, not merely the mechanical procedure, was detrimental to early zygote development and may be the cause of low pregnancy rates.
Enzyme- Facilitated Transport And Separation Of Organic Acids Through Liquid Membranes, David C. Rethwisch, Anuradha Subramanian, Gao Yi, Jonathan S. Dordick
Enzyme- Facilitated Transport And Separation Of Organic Acids Through Liquid Membranes, David C. Rethwisch, Anuradha Subramanian, Gao Yi, Jonathan S. Dordick
Papers in Biotechnology
The selective separation of organic acids is important for the production of numerous biologially functional rnuleculcs, including amino acids, peptidcs, fats, and pharmaceuticals.' Enzyme, specificalty lipases and proteases, have been used to selectively separate a variety of organic acids; however, multiple steps are usually required,Vn additiun, carbonic anhydrase has been usd in a variety of Eiquid membranes for transport of CO2.) In the present study, we have coupled the selectiviey of enzymes with liquid membranes to provide a single-step method to scicctiveiy separate and purify organic acids.
Conformational Changes In An Epitope Localized To The Nh2-Terminal Region Of Protein C, Carolyn L. Orthener, Rapti D. Madurawe, William H. Velander, William N. Drohan, Frances D. Bettey, Dudley K. Strickhand
Conformational Changes In An Epitope Localized To The Nh2-Terminal Region Of Protein C, Carolyn L. Orthener, Rapti D. Madurawe, William H. Velander, William N. Drohan, Frances D. Bettey, Dudley K. Strickhand
Papers in Biotechnology
Murine monoclonal antibodies, developed following immunization with human protein C, were characterized for their ability tboin d antigen in thpe resence of either CaClz or excess EDTA. Three stablec lones were obtained which produced antiboditehsa t bound to protein C only itnh e presence of EDTA. Allt hree antibodies bound to the light choafi np rotein C on immunoblots and also bound to the homologous proteins factor X and prothrombin in solid-phase radioimmunoassays. One antibody, 7D7B10 was purified and studied further. The binding of 7D7B10 to human protein C was characterized bya KOo f 1.4 nM. In competition studies, it …