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At least 523 records · Page 29Linked to original sources
PubMed2000

Effects of yeast expressed recombinant interleukin-2 and interferon-gamma on physiological changes in bovine mammary glands and on bactericidal activity of neutrophils.

The physiological effects of intramammary infusions of recombinant bovine cytokines in six lactating dairy cows on the quality and yield of milk and the bactericidal activity of milk neutrophils were investigated. Recombinant bovine interleukin-2 (rboIL-2) and interferon-gamma (rboIFN-gamma) were produced in the yeast Pichia pastoris. Two animals were given rboIL-2 (2 x 10(5) units) in two quarters, two animals were given rboIFN-gamma (6.5 x 10(5) units) in two quarters, and the other two cows received a dose of rboIL-2 in one quarter and rboIFN-gamma in a second quarter. In addition, each animal was given phosphate-buffered saline (PBS) in the other two quarters as a control. Somatic cell counts and conductivity of the fore milk were monitored before and after infusion. Neutrophils were isolated from quarter milk samples 36 h after infusion of cytokine or PBS and their bactericidal activities against Staphylococcus aureus were measured in vitro with a colorimetric assay. Quarters infused with rboIL-2 or rboIFN-gamma showed significant but transitory increases in both milk somatic cell counts and conductivity when compared with preinfusion values and with control quarters. There were minimal effects on daily milk yield. Neutrophils isolated from milk from quarters infused with rboIL-2 showed enhanced bactericidal activity against Staph. aureus. The bacterial killing from rboIL-2 treated quarters was significantly greater, with a mean of 63.5% compared with a mean of 5.4% for neutrophils taken from uninfected quarters to which PBS had been administered. The bactericidal activities for quarters treated with rboIFN-gamma and infected quarters treated with PBS were 15.0 and 30.0% respectively. The results indicate that intramammary infusions of rboIL-2 and rboIFN-gamma to lactating cows are well tolerated, and that rboIL-2 can activate milk neutrophils and augment their bactericidal activity.

Animals↗
PubMed2004

Probing lipase/esterase libraries for lipid A hydrolases--discovery of biocatalysts for the detoxification of bacterially-expressed recombinant protein.

In our ongoing efforts to develop new methods for lipopolysaccharide (LPS) detoxification, we have screened lipase/esterase libraries for the ability to deacylate the 2'- and 3'-fatty acid chains from lipid A: the most active esterases were successfully employed to inactivate LPSs in a crude concentrated cell supernatant of E. Coli containing a recombinant single chain antibody (scFv).

Animals↗
PubMed2001

Stable recombinant expression and characterization of the two haemophilic factor VIII variants C329S (CRM(-)) and G1948D (CRM(r)).

In haemophilia A, the functional defect at the molecular level of most factor VIII (FVIII) missense mutations remains unknown. Site-directed mutagenesis of B domain-deleted FVIII cDNA (FVIIISQ) was used to introduce two mutations associated with severe cross-reacting material (CRM)-negative (FVIII-C329S) or mild/moderate CRM-reduced (FVIII-G1948D) haemophilia A. Wild-type (FVIIISQ-WT) and variant FVIIISQ proteins were successfully expressed after stable transfection in Chinese hamster ovary (CHO) cells, and partially characterized at the intracellular, molecular and functional levels. Reverse transcription polymerase chain reaction analysis confirmed that both transcription and mRNA processing appeared normal in CHO cells transfected with both the wild-type and two variant constructs. In contrast to FVIIISQ-WT, immunofluorescence analysis of both CRM(-) and CRM(r) variants showed intracellular FVIII accumulation within the rough endoplasmic reticulum, suggesting secretion defects in transfected CHO cells. Immunoblot analysis of the FVIIISQ variant proteins that were secreted showed that they were expressed as mixed populations of uncleaved 170 kDa polypeptides, processed 90 kDa heavy chains and 80 kDa light chains, similar to FVIIISQ-WT. Phenotypic analysis of the B domain-deleted FVIIISQ variants expressed in CHO cells correlated well with the patients' reduced FVIII activity and, in addition, surface plasmon resonance studies demonstrated that both missense mutations were associated with increased rates of A2 domain dissociation following thrombin activation. We conclude that the mutations found are responsible for the haemophilia A phenotype, through intracellular retention and decreased stability of the active cofactor FVIIIa.

Animals↗
PubMed1989

Dual affinity fusion approach and its use to express recombinant human insulin-like growth factor II.

A dual affinity fusion concept has been developed in which the gene encoding the desired product is fused between two flanking heterologous genes encoding IgG- and albumin-binding domains. Using sequential IgG and serum albumin affinity chromatography, a full-length tripartite fusion protein is obtained. This approach was used to recover a full-length fusion product in Escherichia coli containing the human insulin-like growth factor II (IGF-II). Surprisingly, the recombinant IGF-II showed increased stability against proteolytic degradation in E. coli when produced as a dual affinity fusion protein, as compared to an N-terminal fusion protein. After site-specific cleavage of the tripartite fusion protein, IGF-II molecules with immunological and receptor binding activity were obtained without renaturation steps. The results demonstrate that proteins can fold into biologically active structures, even if provided with large flanking heterologous protein domains. The concept was further used to characterize the specific degradation of recombinant IGF-II in this heterologous host.

Amino Acid Sequence↗
PubMed2006

Recombinant expression and biochemical characterization of the unique elongating beta-ketoacyl-acyl carrier protein synthase involved in fatty acid biosynthesis of Plasmodium falciparum using natural and artificial substrates.

The human malaria parasite Plasmodium falciparum synthesizes fatty acids by using a type II synthase that is structurally different from the type I system found in eukaryotes. Because of this difference and the vital role of fatty acids, the enzymes involved in fatty acid biosynthesis of P. falciparum represent interesting targets for the development of new antimalarial drugs. beta-Ketoacyl-acyl carrier protein (ACP) synthase (PfFabBF), being the only elongating beta-ketoacyl-ACP synthase in P. falciparum, is a potential candidate for inhibition. In this study we present the cloning, expression, purification, and characterization of PfFabBF. Soluble protein was obtained when PfFabBF was expressed as a NusA fusion protein in Escherichia coli BL21(DE3)-CodonPlus-RIL cells under conditions of osmotic stress. The fusion protein was purified by affinity and ion exchange chromatography. Various acyl-P. falciparum acyl carrier protein (PfACP) substrates were tested for their specific activities, and their kinetic parameters were determined. Activity of PfFabBF was highest with C(4:0)- to C(10:0)-acyl-PfACPs and decreased with use of longer chain acyl-PfACPs. Consistent with the fatty acid synthesis profile found in the parasite cell, no activity could be detected with C(16:0)-PfACP, indicating that the enzyme is lacking the capability of elongating acyl chains that are longer than 14 carbon atoms. PfFabBF was found to be specific for acyl-PfACPs, and it displayed much lower activities with the corresponding acyl-CoAs. Furthermore, PfFabBF was shown to be sensitive to cerulenin and thiolactomycin, known inhibitors of beta-ketoacyl-ACP synthases. These results represent an important step toward the evaluation of P. falciparum beta-ketoacyl-ACP synthase as a novel antimalaria target.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
PubMed1999

Characterization of the oligosaccharides assembled on the Pichia pastoris-expressed recombinant aspartic protease.

Aspartic protease, widely used as a milk-coagulating agent in industrial cheese production, contains three potential N-glycosylation sites. In this study, we report the characterization of N-linked oligosaccharides on recombinant aspartic protease secreted from the methylotrophic yeast Pichia pastoris using a combination of mass spectrometric, 2D chromatographic, chemical and enzymatic methods. The carbohydrates from site I (Asn79) were found to range from Man6-17GlcNAc2 with 50% bearing a phospho-diester-motif, site II (Asn113) was not occupied and site III (Asn188) contained mostly uncharged species ranging from Man-13GlcNAc2. These charged groups are not affecting the transport through the secretion pathway of the recombinant glycoprotein. Changes from a molasses-based medium to a minimal salts-based medium led to a clear reduction of the degree of phosphorylation of the N-glycan population.

Amino Acid Sequence↗
PubMed1999

Gemcitabine transport in xenopus oocytes expressing recombinant plasma membrane mammalian nucleoside transporters.

BACKGROUND: Gemcitabine, a pyrimidine analogue of deoxycytidine, is an anticancer nucleoside drug that requires functional plasma membrane nucleoside transporter proteins to reach its intracellular targets and cause cytotoxicity. Because of technical difficulties inherent in studying nucleoside transport in human cells, we rigorously defined gemcitabine membrane transportability by producing each of the available human (h) and rat (r) recombinant nucleoside transporters (NTs) individually in Xenopus laevis oocytes. METHODS: Oocytes were microinjected with in vitro-transcribed RNAs derived from complementary DNAs encoding (C = concentrative) rCNT1, rCNT2, hCNT1, hCNT2, (E = equilibrative) rENT1, rENT2, hENT1, and hENT2. Uptake of [(3)H]gemcitabine and [(14)C] uridine was measured 3 days after microinjection to determine kinetic constants. We also used the two-electrode, voltage-clamp technique to investigate the electrophysiology of hCNT1-mediated gemcitabine transport. RESULTS: Gemcitabine was transported by most of the tested proteins (the exceptions being the purine-selective rCNT2 and hCNT2), with the greatest uptake occurring in oocytes producing recombinant rCNT1 and hCNT1. Influxes of gemcitabine mediated by hCNT1, hENT1, and hENT2 were saturable and conformed to Michaelis-Menten kinetics with apparent K(m) values of 24, 160, and 740 microM, respectively. Gemcitabine had a limited ability to cross the lipid bilayer of oocyte membranes by simple diffusion. External application of gemcitabine to oocytes producing recombinant hCNT1 induced an inward current, which demonstrated that hCNT1 functions as a Na(+)/nucleoside co-transport protein and confirmed the transporter's ability to transport gemcitabine. CONCLUSIONS: Mammalian nucleoside transporters vary widely in their affinity and capacity to transport gemcitabine. Variation in the tumor and tissue distribution of plasma membrane nucleoside transporter proteins may contribute to the solid tumor activities and schedule-dependent toxic effects of gemcitabine.

Animals↗
PubMed1996

Enhanced in vivo antithrombotic effects of endothelial cells expressing recombinant plasminogen activators transduced with retroviral vectors.

BACKGROUND: The effects of regulating endothelial cell (EC) plasminogen activator production on thrombus accumulation in vivo are incompletely understood. By overexpressing plasminogen activators in ECs via gene transfer, the hypothesis was tested that increased levels of plasminogen activators inhibit the accumulation of thrombus in vivo. METHODS AND RESULTS: Cultured baboon ECs transduced with human cDNAs for wild-type tissue plasminogen activator (TPA) or for glycosylphosphatidylinositol-anchored urokinase-type plasminogen activator (a-UPA) were seeded onto collagen-coated segments of vascular graft (collagen segments) and exposed overnight to flow using an in vitro perfusion circuit. The antigenic levels of TPA and UPA each increased 10-fold in the media perfusing the corresponding transduced ECs compared with untransduced ECs (P < or = .05 in both cases). In baboons the antithrombotic effects of TPA-transduced or a-UPA-transduced ECs were measured as 111In-platelet deposition and 125I-fibrin accumulation on collagen segments bearing sparsely attached ECs (tarnsduced versus untransduced) interposed in exteriorized arteriovenous femoral shunts. Platelet-rich thrombus formed on the collagen segments with fibrin-rich thrombus propagated distally. The presence of TPA-transduced or a-UPA-transduced ECs on collagen segments at a density of 25,000 ECs/cm2 decreased 111AIn-platelet deposition and 125I-fibrin accumulation on collagen surfaces compared with untransduced ECs present at equivalent density (P < .05 for platelet deposition with TPA-transduced ECs and P < .05 for platelet deposition on the propagated tail, as well as fibrin accumulation on the graft with a-UPA-transduced ECs). The systemic levels of fibrinopeptide A, thrombin-antithrombin complex, D-dimer, and both local and systemic levels of TPA and UPA were not increased by transduced ECs compared with untransduced ECs. The focal antithrombotic effects of transduced ECs appear to be due to local enhancement of thrombolysis. CONCLUSIONS: ECs transduced with recombinant TPA and a-UPA enhance local antithrombotic activity in vivo. This strategy of attaching transduced ECs overexpressing plasminogen activators may be therapeutically useful by preventing thrombo-occlusive failure of implanted cardiovascular devices or mechanically denuded vessels.

Animals↗
PubMed2001

Specific inhibitors prevent proteolytic degradation of recombinant proteins expressed in High Five cells.

The insect cell line BTI-TN-5B1-4 (High Five) is frequently used to express recombinant proteins in large amounts using the baculovirus expression system. However, extensive proteolytic degradation of recombinant proteins is often encountered. Furthermore, we have observed that recombinant proteins migrate in SDS-PAGE in agreement with poly-ubiquitinated forms of the protein, suggesting a ubiquitin/proteasome degradation pathway. Here, we describe a systematic study unraveling the effect of adding proteasome inhibitors or specific protease inhibitors to the growth medium of High Five insect cells infected with recombinant baculovirus. Furthermore, protease inhibitors were added to the lysis buffer to establish the most efficient way to inhibit proteolytic activity after lysis of baculovirus-infected cells expressing recombinant proteins. We conclude that a combination of adding protease inhibitors to the growth medium and to the lysis buffer minimizes the proteolytic activity in High Five cells. The most efficient protease inhibitors were E-64 in the growth medium together with Leupeptin in the lysis buffer at concentrations higher than with available cocktails of inhibitors. The optimal treatment of High Five cells is different from the optimal treatment of Sf9 cells. For proteins susceptible to ubiquitinylation, a treatment of insect cell cultures with the proteasome inhibitor MG132 (LLL) leads to a considerable reduction of the yield of production of recombinant protein.

Animals↗
PubMed1997

Enhanced Bioactivity of Recombinant Baculoviruses Expressing Insect-Specific Spider Toxins in Lepidopteran Crop Pests

Two genetically enhanced isolates of the Autographa californica nuclear polyhedrosis virus (AcMNPV) expressing insect-specific neurotoxin genes from the spiders Diguetia canities and Tegenaria agrestis were evaluated for their commercial potential. Since prevention of feeding damage is of primary importance in assessing agronomic efficacy, a method for estimating the median time to cessation of feeding (FT50) was developed. Neonate droplet feeding assays with preoccluded virus samples were conducted to compare the FT50s and median survival times (ST50s) of larvae infected by the toxin-expressing recombinant viruses with those of larvae infected by wild-type AcMNPV and the appropriate polyhedrin-minus control viruses. Low dosages were used to minimize the effect of dosage on the response times, and the time to molting of noninfected larvae was used to audit variability among batches of larvae within and between tests. Appropriate statistics are discussed. To evaluate host spectrum, response times were compared in three lepidopteran insect pests, Trichoplusia ni Hubner, Spodoptera exigua (Hubner), and Heliothis virescens (Fabricius). The recombinant viruses expressing insect-specific toxin genes from T. agrestis and D. canities, designated vAcTalTX-1 and vAcDTX9.2, respectively, significantly reduced both FT50 and ST50 in all three lepidopteran pests. Reductions in feeding times compared to the wild-type virus ranged from 16 to 39% with vAcTalTX-1 and 30 to 40% with vAcDTX9.2. Reductions in survi val time were lower, ranging from 18 to 33% with vAcTalTX-1 and 9 to 24% with vAcDTX9.2. While vAcTalTX-1 tended to kill faster than vAcDTX9.2, vAcDTX9.2 stopped feeding faster than vAcTalTX-1, suggesting that it would be more effective in reducing crop damage.

Journal Article↗
PubMedDate not supplied

Hepatitis B virus core-preS2 particles expressed by recombinant vaccinia virus.

Vaccinia virus (VV) recombinants expressing hepatitis B virus (HBV) surface (HBsAg) or core (HBcAg) antigens (Kunke et al., Virology 195, 132 - 139 (1993)] have been shown to raise specific antibodies in mice, nevertheless the levels of antibodies reactive with the preS2 and S antigens were low. In an attempt to enhance the immunogenicity of HBsAg-preS2, a fused C-preS2 gene was constructed. The fusion protein was expressed in E. coli and displayed both HBcAg and preS2 antigen as demonstrated by enzyme-linked immunosorbent assay (ELISA). The same gene was then expressed using recombinant VV and chimerical particles whose size and density were similar to those of native HBV core particles produced in CV-1 cells infected with recombinant VV. Unlike HBcAg, preS2 antigen could not be detected on these particles by ELISA but was revealed by immunoblot analysis only. The immunogenicity of the recombinant VV was evaluated in mice. Antibodies to HBcAg and VV antigen but not to preS2 antigen were found in sera of animals inoculated with 10(7) PFU of the recombinant VV. Presumably, HBcAg-preS2 particles produced in E. coli and in eukaryotic cells have a different conformation, and the presence of preS2 antigen on the surface of chimerical particle might be necessary for a pronounced antibody response.

Animals↗