Open Access. Powered by Scholars. Published by Universities.®

Biochemical and Biomolecular Engineering Commons™

Open Access. Powered by Scholars. Published by Universities.®

University of Nebraska - Lincoln

Discipline
Keyword
Publication Year
Publication
File Type

Articles 91 - 120 of 125

Full-Text Articles in Biochemical and Biomolecular Engineering

Causes Of Proteolytic Degradation Of Secreted Recombinant Proteins Produced In Methylotrophic Yeast Pichia Pastoris: Case Study With Recombinant Ovine Interferon-Τ, Mehmet İnan, Michael M. Meagher, Bradley A. Plantz, Jayanta Sinha Mar 2004

Causes Of Proteolytic Degradation Of Secreted Recombinant Proteins Produced In Methylotrophic Yeast Pichia Pastoris: Case Study With Recombinant Ovine Interferon-Τ, Mehmet İnan, Michael M. Meagher, Bradley A. Plantz, Jayanta Sinha

Papers in Biochemical Engineering

It was observed that during fermentative production of recombinant ovine interferon-τ (r-oIFN-τ) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in increasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were …

Go to article

Characterization And Optimization Of A Chromatographic Process Based On Ethylenediamine-N,N,N′,N′-Tetra(Methylphosphonic) Acid-Modified Zirconia Particles, Sabyasachi Sarkar, Peter W. Carr, Clayton V. Mcneff, Anuradha Subramanian Jun 2003

Characterization And Optimization Of A Chromatographic Process Based On Ethylenediamine-N,N,N′,N′-Tetra(Methylphosphonic) Acid-Modified Zirconia Particles, Sabyasachi Sarkar, Peter W. Carr, Clayton V. Mcneff, Anuradha Subramanian

Papers in Biotechnology

The primary objective of work was to characterize, optimize and model a chromatographic process based on ethylenediamine-N,N,N′,N′-tetra(methylphosphonic) acid (EDTPA)-modified zirconia particles. Zirconia particles were produced by spray-drying colloidal zirconia. Zirconia spheres produced were further classified, calcined and modified with EDTPA to yield a solid-phase support for use in bio-chromatography (r_PEZ). Specifically, the ability of r_PEZ to selectively bind and enrich IgG, IgA, and IgM from biological fluids was evaluated and demonstrated. To better understand the force of interaction between the IgG and the r_PEZ, the equilibrium disassociation constant (Kd) was determined by static binding isotherms, as a function of temperature …

Go to article

Transgenic Non-Human Mammals Expressing Human Coagulation Factor Viii And Von Willebrand Factor, Henryk Lubon, William N. Drohan, William H. Velander Feb 2003

Transgenic Non-Human Mammals Expressing Human Coagulation Factor Viii And Von Willebrand Factor, Henryk Lubon, William N. Drohan, William H. Velander

Papers in Biotechnology

A non-human transgenic mammalian animal, as described above, contains an exogenous double stranded DNA sequence stably integrated into the genome of the animal, which comprises cis-acting regulatory units operably linked to a DNA sequence encoding human Factor VIII protein and a signal peptide, where the cis-acting regulatory units are active in mammary gland cells and the signal peptide is active in directing newly expressed Factor VIII into the milk of the animal. The promoter may be a milk protein promoter such as for whey acidic protein, casein, lactalbumin, or beta-lactoglobulin promoter. The transgenic mammals are preferably farm animals, for example, …

Go to article

Pichia Pastoris Fermentation With Mixed-Feeds Of Glycerol And Methanol: Growth Kinetics And Production Improvement, Wenhui Zhang, Karen J. Hywood Potter, Bradley A. Plantz, Vicki L. Schlegel, Leonard A. Smith, Michael M. Meagher Jan 2003

Pichia Pastoris Fermentation With Mixed-Feeds Of Glycerol And Methanol: Growth Kinetics And Production Improvement, Wenhui Zhang, Karen J. Hywood Potter, Bradley A. Plantz, Vicki L. Schlegel, Leonard A. Smith, Michael M. Meagher

Papers in Biochemical Engineering

Fed-batch fermentation of a methanol utilization plus (Mut+) Pichia pastoris strain typically has a growth phase followed by a production phase (induction phase). In the growth phase glycerol is usually used as carbon for cell growth while in the production phase methanol serves as both inducer and carbon source for recombinant protein expression. Some researchers employed a mixed glycerol-methanol feeding strategy during the induction phase to improve production, but growth kinetics on glycerol and methanol and the interaction between them were not reported. The objective of this paper is to optimize the mixed feeding strategy based on growth kinetic studies …

Go to article

An Approach To Sequence Dna Without Tagging, Sanjun Niu, Ravi F. Saraf Sep 2002

An Approach To Sequence Dna Without Tagging, Sanjun Niu, Ravi F. Saraf

Papers in Biotechnology

Microarray technology is playing an increasingly important role in biology and medicine and its application to genomics for gene expression analysis has already reached the market with a variety of commercially available instruments. In these combinatorial analysis methods, known probe single-strand DNA (ssDNA) ‘primers’ are attached in clusters of typically 100 μm × 100 μm pixels. Each pixel of the array has a slightly different sequence. On exposure to ‘unknown’ target ssDNA, the pixels with the right complementary probe ssDNA sequence convert to double-stranded DNA (dsDNA) by a hybridization reaction. To transduct the conversion of the pixel to dsDNA, the …

Go to article

Thermodynamics And Bioenergetics, Yaşar Demirel, Stanley I. Sandler Mar 2002

Thermodynamics And Bioenergetics, Yaşar Demirel, Stanley I. Sandler

Papers in Thermal Mechanics

Bioenergetics is concerned with the energy conservation and conversion processes in a living cell, particularly in the inner membrane of the mitochondrion. This review summarizes the role of thermodynamics in understanding the coupling between the chemical reactions and the transport of substances in bioenergetics. Thermodynamics has the advantages of identifying possible pathways, providing a measure of the efficiency of energy conversion, and of the coupling between various processes without requiring a detailed knowledge of the underlying mechanisms. In the last five decades, various new approaches in thermodynamics, non-equilibrium thermodynamics and network thermodynamics have been developed to understand the transport and …

Go to article

Thermodynamics And Bioenergetics, Yaşar Demirel, Stanley I. Sandler Mar 2002

Thermodynamics And Bioenergetics, Yaşar Demirel, Stanley I. Sandler

Papers in Thermal Mechanics

Bioenergetics is concerned with the energy conservation and conversion processes in a living cell, particularly in the inner membrane of the mitochondrion. This review summarizes the role of thermodynamics in understanding the coupling between the chemical reactions and the transport of substances in bioenergetics. Thermodynamics has the advantages of identifying possible pathways, providing a measure of the efficiency of energy conversion, and of the coupling between various processes without requiring a detailed knowledge of the underlying mechanisms. In the last five decades, various new approaches in thermodynamics, non-equilibrium thermodynamics and network thermodynamics have been developed to understand the transport and …

Go to article

Expression Of Active Human Factor Ix In Mammary Tissue And Of Milk Non Human Transgenic Mammals , William H. Velander, William N. Drohan Feb 2002

Expression Of Active Human Factor Ix In Mammary Tissue And Of Milk Non Human Transgenic Mammals , William H. Velander, William N. Drohan

Papers in Biotechnology

Recombinant Factor IX characterized by a high percentage of active protein can be obtained in the milk of transgenic animals that incorporate chimeric DNA molecules according to the present invention. Transgenic animals of the present invention are produced by introducing into developing embryos DNA that encodes Factor IX, such that the foreign DNA is stably incorporated in the DNA of germ line cells of the mature animal. Particularly efficient expression was accomplished using a chimeric construct comprising a mammary gland specific promoter, Factor IX cDNA that lacked the complete or any portion of the 5'-untranslated and 3'-untranslated region, which is …

Go to article

Non-Repressing Carbon Sources For Alcohol Oxidase (Aoxi) Promoter Of Pichia Pastoris, Mehmet İnan, Michael M. Meagher Nov 2001

Non-Repressing Carbon Sources For Alcohol Oxidase (Aoxi) Promoter Of Pichia Pastoris, Mehmet İnan, Michael M. Meagher

Papers in Biochemical Engineering

The growth of Pichia pastoris in a mixture of either glycerol or glucose and methanol follows a diauxic growth, with C1, utilizing enzymes being repressed. Therefore, these carbon sources can not be used as a mixture with methanol to simultaneously grow P. pastoris and induce C1, utilizing enzymes, especially in shake flask cultures of AOX-deficient P. pastoris. Among the alternative carbon sources tested, alanine, sorbitol, mannitol and trehalose, did not repress β-gal production when methanol was used as an inducer in mut- strain of P. pastoris. Our results show that either one of alanine, sorbitol, …

Go to article

Characteristics And Sequence Of Phosphoglycolate Phosphatase From A Eukaryotic Green Alga Chlamydomonas Reinhardtii, Tarlan G. Mamedov, Kensaku Suzuki, Kenji Miura, Ken-Ichi Kucho, Hideya Fukuzawa Oct 2001

Characteristics And Sequence Of Phosphoglycolate Phosphatase From A Eukaryotic Green Alga Chlamydomonas Reinhardtii, Tarlan G. Mamedov, Kensaku Suzuki, Kenji Miura, Ken-Ichi Kucho, Hideya Fukuzawa

Papers in Biochemical Engineering

Phosphoglycolate phosphatase (PGPase), a key en¬zyme of photorespiration in photosynthetic organisms, was purified from Chlamydomonas reinhardtii. The en¬zyme was an ~65-kDa homodimer with a pI value of 5.1 composed of ~32-kDa subunits not connected by any S–S bridges. It was also highly specific for phosphoglycolate with a Km value of 140 μM and an optimal pH between 8 and 9. The activity was strongly inhibited by CaCl2, and it recovered competitively following the addition of MgCl2 or EGTA. A mobility shift was observed in SDS-polyacrylamide gel electrophoresis by the addition of CaCl2, indicating that the enzyme binds to Ca2+ . …

Go to article

The Effect Of Ethanol And Acetate On Protein Expression In Pichia Pastoris, Mehmet İnan, Michael M. Meagher Jul 2001

The Effect Of Ethanol And Acetate On Protein Expression In Pichia Pastoris, Mehmet İnan, Michael M. Meagher

Papers in Biochemical Engineering

Pichia pastoris is an excellent host for high-level heterologous gene expression, but there is still much interest in improving the productivity of recombinant protein production. P. pastoris produces a small amount of ethanol as a by-product during the glycerol fed-batch phase and the mixed-feed induction phase (glycerol-methanol) of high cell density fermentations, regardless of the phenotype (Mutf, Mut" or Mut-). We have investigated ethanol repression of the AOXl promoter using strains, GS115 (Mutf) and MC100-3 (Mut-), expressing an AOXI-lac2 fusion. The addition of 10 mg t1 ethanol at the start of methanol induction delayed P-galactosidase production and methanol utilization …

Go to article

Expression Of A Heterologous Protein C In Mammary Tissue Of Transgenic Animals Using A Long Whey Acidic Protein Promoter , William H. Velander, Henryk Lubton, William N. Drohan, Lothar Hennighausen Jul 2001

Expression Of A Heterologous Protein C In Mammary Tissue Of Transgenic Animals Using A Long Whey Acidic Protein Promoter , William H. Velander, Henryk Lubton, William N. Drohan, Lothar Hennighausen

Papers in Biotechnology

An isolated DNA sequence which regulates the expression of a heterologous gene composed of a mouse whey acidic protein promoter having a length of greater than about 2.4 kb extending upstream from the unique KpnI site in the mouse whey acidic protein gene is disclosed. Specifically a mouse whey acidic protein promoter of about 4.1-4.2 kb in length extending upstream from the unique KpnI site is preferred. This mouse whey acid protein promoter is operably linked to a DNA sequence encoding a heterologous polypeptide and used to prepare transgenic non-human mammals expressing the heterologous polypeptide in their milk. Particularly efficient …

Go to article

Modeling Pichia Pastoris Growth On Methanol And Optimizing The Production Of A Recombinant Protein, The Heavy-Chain Fragment C Of Botulinum Neurotoxin, Serotype A, Wenhui Zhang, Mark A. Bevins, Bradley A. Plantz, Leonard A. Smith, Michael M. Meagher Oct 2000

Modeling Pichia Pastoris Growth On Methanol And Optimizing The Production Of A Recombinant Protein, The Heavy-Chain Fragment C Of Botulinum Neurotoxin, Serotype A, Wenhui Zhang, Mark A. Bevins, Bradley A. Plantz, Leonard A. Smith, Michael M. Meagher

Papers in Biotechnology

An unstructured growth model for the recombinant methylotrophic yeast P. pastoris Mut+ expressing the heavy-chain fragment C of botulinum neurotoxin serotype A [BoNT/A(Hc)], was successfully established in quasi-steady state fed-batch fermentations with varying cell densities. The model describes the relationships between specific growth rate and methanol concentration, and the relationships between specific methanol and ammonium consump-tion rates and specific growth rate under methanol-limited growth conditions. The maximum specific growth rate (μ) determined from the model was 0.08 h−1 at a methanol concentration of 3.65 g/L, while the actual maximum μ was 0.0709 h−1. The maximum specific methanol consumption rate was …

Go to article

Fermentation Strategies For Recombinant Protein Expression In The Methylotrophic Yeast Pichia Pastoris, Wenhui Zhang, Mehmet Inan, Michael M. Meagher Aug 2000

Fermentation Strategies For Recombinant Protein Expression In The Methylotrophic Yeast Pichia Pastoris, Wenhui Zhang, Mehmet Inan, Michael M. Meagher

Papers in Biochemical Engineering

Fermentation strategies for recombinant protein production in Pichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, application of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.

Go to article

Immunoaffinity Chomatography, Anuradha Subramanian Apr 2000

Immunoaffinity Chomatography, Anuradha Subramanian

Papers in Biotechnology

Recent devclopments in recombinant DNA technology have enabled the synthesis of valuable therapeutic proteins in bacterial cells as well as in novel eucaryotic expression systems. However, the purification of proteins of interest from either the conventional sources, cell culture, or novel routes in a highly purified form necessitates the development of separation tcchniques capable of recovering proteins from these feed streams in a highly purified form (1,2). Purification of therapeutic proteins from biological sources is usually complicated by the presence of endogenous proteins (2). Purification methodologies based on ion exchange or adsorption serve as excellent prepurification steps, but they fail …

Go to article

Purification Of Immunoglobulins From Serum Using Thiophilic Cellulose Beads, Anuradha Subramanian Jan 2000

Purification Of Immunoglobulins From Serum Using Thiophilic Cellulose Beads, Anuradha Subramanian

Papers in Biotechnology

This study evaluates the chromatographic performance of a support obtained by the reaction of mercaptoethanol with cellulose beads activated with divinyl sulfone. Cellulose beads 500-800 microns (pm) in diameter and with a solids content of 3.5% were selected for this study. A two-step sequence of permeation and reaction was used to install thiophilic sites throughout the cross section of the bead. The distribution of thiophilic sites was visualized by immobilizing fluorescent antibodies. Human and porcine serum proteins were separated on the thiophilic support at different linear velocities. Thiophilic cellulose beads were observed to bind human and porcine immunoglobulins (IgG) selectively …

Go to article

Ranking The Factors Impacting Lmmunosorbent Performance, Anuradha Subramanian, William H. Velander Jan 2000

Ranking The Factors Impacting Lmmunosorbent Performance, Anuradha Subramanian, William H. Velander

Papers in Biotechnology

This work addresses the relative impact of the phenomena of orientation, multipoint attachment, and local density of the immobilized antibody 011 immunosorbent perfornnance. The masking of the antigen binding domains of monoclonal antibodies (Mab) by Fab Masking Antigens (FMAs) was used as a tool to determine the impact of orientation on the perforn~anceo f Emphaze, Affiprep'M, and Cellulose bead-based immunosorbents. A two-step antibody immobilization inethod involving permeation of the beaded matrix followed by a fast coupling reaction was used to study the effect of local density of the Mnh on immunosorbent efficiency. Lysyl groups on Mabs were covalently modified to …

Go to article

Bioactivities Of A Tumour Necrosis Like Factor Released By Chicken Rnacrophages, Silke Rautenschlein, Anuradha Subramanian, Jagdev M. Sharma Jul 1999

Bioactivities Of A Tumour Necrosis Like Factor Released By Chicken Rnacrophages, Silke Rautenschlein, Anuradha Subramanian, Jagdev M. Sharma

Papers in Biotechnology

To test for tumour necrosis-like factor (TNF) of chickens, supernatants of a lipopolysaccharide (LPS)-stimulated chicken macrophage cell line MQ-NCSU were analysed. A sequence of ion-exchange and gel-permeation chromatography was utilised to isolate TNF-like activity from the culture supernatant. The peak of TNF-like cytotoxic activity corresponded to the fractions with a nlolecular weight of 81 kDa or higher. Polyclonal anti-human TNF-a antiserum cross-reacted by Western blotting with a 17 kDa protein in the TNF-containing fraction ~ ~ n d e denaturing conditions. This result indicated that chicken TNF-like factor in the biologically active form may be a protein multimer of monomers …

Go to article

Bioactivities Of A Tumour Necrosis Like Factor Released By Chicken Macrophages, Silke Rautenschlein, Anuradha Subramanian, Jagdev M. Sharma Jul 1999

Bioactivities Of A Tumour Necrosis Like Factor Released By Chicken Macrophages, Silke Rautenschlein, Anuradha Subramanian, Jagdev M. Sharma

Papers in Biotechnology

To test for tumour necrosis-like factor (TNF) of chickens, supernatants of a lipopolysaccharide (LPS)-stimulated chicken macrophage cell line MQ-NCSU were analysed. A sequence of ion-exchange and gel-permeation chromatography was utilised to isolate TNF-like activity from the culture supernatant. The peak of TNF-like cytotoxic activity corresponded to the fractions with a nlolecular weight of 81 kDa or higher. Polyclonal anti-human TNF-a antiserum cross-reacted by Western blotting with a 17 kDa protein in the TNF-containing fraction ~ ~ n d e denaturing conditions. This result indicated that chicken TNF-like factor in the biologically active form may be a protein multimer of monomers …

Go to article

Biodegradation Of Thermoplastic And Hermosetting Polyesters From Z-Protected Glutamic Acid, R M. Tadros, Hossein Noureddini, Delmar C. Timm Jun 1999

Biodegradation Of Thermoplastic And Hermosetting Polyesters From Z-Protected Glutamic Acid, R M. Tadros, Hossein Noureddini, Delmar C. Timm

Papers in Biomolecular Engineering

In a previous article,1 the development and molecular characterization of three polyesters from N-carbobenzyloxy-L-glutamic acid (ZGluOH) were reported. The polymers were a linear, heterochain polyester (ZGluOH and ethylene glycol), a crosslinked heterochain polyester (ZGluOH and diglycidyl ether of 1,4-butanediol), and a crosslinked, heterochain aromatic polyester (ZGluOH and diglycidyl ether of bisphenol A). In this manuscript, results of biodegradation studies are reported. The three polymers hydrolyzed to low molecular weight oligomers similar to the monomers with lipase. When exposed to a mixed culture of micro-organisms, the first two resins degraded to biomass and respiratory gases. The crosslinked heterochain aromatic polyester resisted …

Go to article

A Mutant Of Chlamydomonas Reinhardtii With Reduced Rate Of Photorespiration, Kensaku Suzuki, Tarlan G. Mamedov, Toraoyoshi Ikawa Feb 1999

A Mutant Of Chlamydomonas Reinhardtii With Reduced Rate Of Photorespiration, Kensaku Suzuki, Tarlan G. Mamedov, Toraoyoshi Ikawa

Papers in Biochemical Engineering

Photorespiration rates under air-equilibrated conditions (0.04% CO2 and 21% O2) were measured in Chlamydomonas reinhardtii wild-type 2137, a phosphoglycolate-phosphatase-deficient (pgpl) mutant and a suppressor double mutant (7FR2N) derived from the pgpl mutant. In both cells grown under 5% CO2 and adapted air for 24 h in the suppressor double mutant, the maximal rate of photorespiration (phosphoglycolate synthesis) was only about half of that in either the wild type or the pgpl mutant (18-7F) cells. In the progeny, the reduced rate of photorespiration was accompanied by increased photosynthetic affinity for inorganic carbon and the capacity for growth …

Go to article

Bovine Follicular Dynamics, Oocyte Recovery,And Development Of Oocytes Microinjected With A Green Fluorescent Protein Construct, M S. Chauhan, S Nadir, T L. Bailey, A W. Pryor, S P. Butler, D R. Notter, William H. Velander, F C. Gwazdauskas Jan 1999

Bovine Follicular Dynamics, Oocyte Recovery,And Development Of Oocytes Microinjected With A Green Fluorescent Protein Construct, M S. Chauhan, S Nadir, T L. Bailey, A W. Pryor, S P. Butler, D R. Notter, William H. Velander, F C. Gwazdauskas

Papers in Biotechnology

The present study was carried out to 1) evaluate the viability of in vitro fertilized zygotes after microinjection of DNA, 2) assess the influence of oocyte quality upon the development rate of embryos when injected with DNA, and 3) determine the integration frequency of green fluorescent protein DNA into microinjected embryos. Oocytes were aspirated from ovaries of nine nonlactating Holsteins and were categorized into grades A, B, C, and D. At 16 h after in vitro fertilization, approximately half of the pronuclear stage presumptive zygotes were classified as having 1 pronucleus or 2 pronuclei, and they were microinjected with DNA …

Go to article

A Red-Dot-Blot Protein Assay Technique In The Low Nanogram Range, Tiilin Marcol, Anuradha Subramanian Jan 1999

A Red-Dot-Blot Protein Assay Technique In The Low Nanogram Range, Tiilin Marcol, Anuradha Subramanian

Papers in Biotechnology

A simple, sensitive, rapid (3 min), and highly reproducible solid-phase assay for the detection of proteins in the low nanogram range (4 ng) is described. The assay is based on differential Pon-ceau S staining of the protein spots on nitrocellulose and quantification of the protein- dye com-plexes on lubricated membranes using a densitometer. The dye solution used for protein stain-ing contained 0.1% Ponceau S in 15% phosphoric acid and 10% ethanol. Proteins were directly spotted onto pre- Ponceau S-stained nitrocellulose membranes, crosslinked with glutaraldehyde, rinsed in NaOH, restained with Ponceau S, and finished by rinsing in acid water at pH …

Go to article

A Red-Dot-Blot Protein Assay Technique In The Low Nanogram Range, Tiilin Marcol, Anuradha Subramanian Jan 1999

A Red-Dot-Blot Protein Assay Technique In The Low Nanogram Range, Tiilin Marcol, Anuradha Subramanian

Papers in Biotechnology

A simple, sensitive, rapid (3 min), and highly reproducible solid-phase assay for the detection of proteins in the low nanogram range (4 ng) is described. The assay is based on differential Pon-ceau S staining of the protein spots on nitrocellulose and quantification of the protein- dye com-plexes on lubricated membranes using a densitometer. The dye solution used for protein stain-ing contained 0.1% Ponceau S in 15% phosphoric acid and 10% ethanol. Proteins were directly spotted onto pre- Ponceau S-stained nitrocellulose membranes, crosslinked with glutaraldehyde, rinsed in NaOH, restained with Ponceau S, and finished by rinsing in acid water at pH …

Go to article

Optimization Of Temperature-Glycerol-Ph Conditions For Fed-Batch Fermentation Process For Recombinant Hookworm (Ancylostoma Caninum) Anticoagulant Peptide (Acap-5) Production By Pichia Pastoris, Mehmet İnan, Vijay Chiruvolu, Kent M. Eskridge, George P. Vlasuk, Kenneth Dickerson, Scott Brown, Michael M. Meagher Dec 1998

Optimization Of Temperature-Glycerol-Ph Conditions For Fed-Batch Fermentation Process For Recombinant Hookworm (Ancylostoma Caninum) Anticoagulant Peptide (Acap-5) Production By Pichia Pastoris, Mehmet İnan, Vijay Chiruvolu, Kent M. Eskridge, George P. Vlasuk, Kenneth Dickerson, Scott Brown, Michael M. Meagher

Papers in Biochemical Engineering

This study was undertaken to determine the optimum pH, temperature and glycerol feed rate for the production of recombinant hookworm (Ancylostoma caninum) anticoagulant peptide (rAcAP-5) by Pichia pastoris using response surface methodology (RSM). A central composite design was used as an experimental design for allocation of treatment combinations in three blocks. The variables selected for study were pH, temperature and glycerol feed rate. pH was the most important variable affecting yield, specific yield and specific activity of rAcAP-5. Glycerol feed rate had a significant effect on the specific activity of rAcAP-5 (% of total secreted protein) while temperature …

Go to article

Crossflow Microfiltration Of Recombinant Escherichia Coli Lysates After High Pressure Homogenization, Stuart M. Bailey, Michael M. Meagher Nov 1997

Crossflow Microfiltration Of Recombinant Escherichia Coli Lysates After High Pressure Homogenization, Stuart M. Bailey, Michael M. Meagher

Papers in Biotechnology

Abstract: Crossflow membrane filtration was used to process recombinant Escherichia coli cell lysates containing protein inclusion bodies after high pressure homogenization. The number of passes through the high pressure homogenizer changed the viscosities and average particle sizes of the cell lysates. The different cell lysates were processed with a hollow fiber unit containing microfiltration membranes and a plate and frame unit with either ultrafiltration or microfiltration membranes. There were differences in permeate flux and protein transmission for the various membranes with the best performing membranes giving permeate fluxes greater than 60 L m−2 h−1 and protein transmissions greater than 90%. …

Go to article

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 Sep 1996

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 …

Go to article

Effect Of Antibody Orientation On Immunosorbent, Anuradha Subramanian, William H. Velander Sep 1996

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. …

Go to article

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 Nov 1994

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 …

Go to article

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 May 1994

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. …

Go to article

First Prev Next Last