PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Recombinant expression”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources
PubMed1992

Immunity in strain 2 guinea-pigs inoculated with vaccinia virus recombinants expressing varicella-zoster virus glycoproteins I, IV, V or the protein product of the immediate early gene 62.

The immunogenicity of specific varicella-zoster virus (VZV) proteins, with emphasis upon cell-mediated immune responses, was evaluated by immunizing strain 2 guinea-pigs with vaccinia virus recombinants that express gpI (vac-gpI), gpIV (vac-gpIV) and gpV (vac-gpV) or the IE-62 protein (vac-IE-62). Vac-gpI elicited the highest initial mean T cell proliferation response [stimulation index (S.I.) 3.8 +/- 0.9 S.E.M.] whereas inoculation with vac-gpV produced the lowest primary T cell response (S.I. 2.5 +/- 1.1 S.E.M.). T cell proliferation was detected for a shorter period after immunization with vac-gpV compared to vac-gpI, vac-gpIV or vac-IE-62. A comparison of the immunogenicity of vac-gpI and vac-IE-62 with the same proteins prepared by immunoaffinity purification showed that immunization with these proteins in either form elicited virus-specific IgG antibodies and T cell recognition. The presence or absence of IgG antibodies to the IE-62 protein was used to assess protection against challenge with guinea-pig cell-adapted infectious VZV in animals that had been inoculated with vac-gpI, vac-gpIV or vac-gpV. Immunization with vac-gpI and vac-gpIV restricted VZV replication but all animals given vac-gpV developed antibodies to IE-62 after challenge with infectious VZV. Priming of the T lymphocyte response was observed in all animals immunized with VZV-vaccinia virus recombinants after subsequent exposure to infectious VZV. These experiments with VZV vac-gpI, vac-gpIV and vac-gpV in guinea-pigs suggest variability in the capacity of herpesviral glycoproteins to elicit cell-mediated immunity in vivo. Induction of virus-specific immunity using IE-62 means that this major tegument protein of VZV could be a useful component for vaccine development.

Animals↗
PubMed1985

Vaccinia virus recombinants: expression of VSV genes and protective immunization of mice and cattle.

Vesicular stomatitis virus (VSV) causes a contagious disease of horses, cattle, and pigs. When DNA copies of messenger RNA's for the G or N proteins of VSV were linked to a vaccinia virus promoter and inserted into the vaccinia genome, the recombinants retained infectivity and synthesized VSV polypeptides. After intradermal vaccination with live recombinant virus expressing the G protein, mice produced VSV-neutralizing antibodies and were protected against lethal encephalitis upon intravenous challenge with VSV. In cattle, the degree of protection against intradermalingually injected VSV was correlated with the level of neutralizing antibody produced following vaccination.

Animals↗
PubMed2001

Protective immune response against cutaneous leishmaniasis by prime/booster immunization regimens with vaccinia virus recombinants expressing Leishmania infantum p36/LACK and IL-12 in combination with purified p36.

In susceptible mice Leishmania infection triggers a CD4(+) Th2 response that has been correlated with evasion of the host immune system. To develop approaches that might trigger a Th1 response leading to protection against Leishmania we generated vaccinia virus recombinants (VVr) expressing the relevant p36/LACK protein of Leishmania infantum (VVp36) or co-expressing p36/LACK and interleukin-12 (VVp36IL12). Susceptible BALB/c mice were immunized with the VVr in various prime/booster protocols that included purified p36/LACK protein, followed 3 weeks later by a challenge with live L. major promastigotes. The course of the infection was monitored by measuring lesion development, parasite load and immunological parameters (IFN-gamma and IL-10 secretion by in vitro-stimulated lymphocytes, and specific IgG isotypes), before and after challenge. We found protocols of prime/booster immunization (VVp36/VVp36; VVp36IL12/p36; p36/VVp36IL12) that elicited different levels of protection in infected animals. The protocol of priming with purified p36 followed by a booster with VVp36IL12 induced 52% reduction in lesion size and a two-log unit reduction in parasite load. This partial protection correlated with activation of a specific Th1 type of immune response. These protocols could be of interest in the prophylaxis against Leishmania spp. and other parasitic diseases.

Animals↗
PubMed1998

Novel aspects of tetramer assembly and N-terminal domain structure and function are revealed by recombinant expression of human AMP deaminase isoforms.

AMP deaminase isoforms purified from endogenous sources display smaller than predicted subunit molecular masses, whereas baculoviral expression of human AMPD1 (isoform M) and AMPD3 (isoform E) cDNAs produces full-sized recombinant enzymes. However, nearly 100 N-terminal amino acid residues are cleaved from each recombinant polypeptide during storage at 4 degreesC. Expression of N-truncated cDNAs (DeltaL96AMPD1 and DeltaM90AMPD3) produces stable recombinant enzymes exhibiting subunit molecular masses and kinetic properties that are similar to those reported for purified isoforms M and E. Conversely, wild type recombinant isoforms display significantly higher Km(app) values in the absence of ATP. Gel filtration analysis demonstrates native tetrameric structures for all recombinant proteins, except the wild type AMPD1 enzyme, which forms aggregates of tetramers that disperse upon cleavage of N-terminal residues at 4 degreesC. These data: 1) confirm that available literature on AMP deaminase is likely derived from N-truncated enzymes and 2) are inconsistent with a new model proposing native trimeric structure of an N-truncated rabbit skeletal muscle AMP deaminase (Ranieri-Raggi, M., Montali, U., Ronca, F., Sabbatini, A., Brown, P. E., Moir, A. J. G., and Raggi, A. (1997) Biochem. J. 326, 641-648). N-terminal residues also influence actomyosin-binding properties of the enzyme, which are enhanced and suppressed by AMPD1 and AMPD3 sequences, respectively. Finally, co-expression of AMPD1 and AMPD3 recombinant polypeptides produces tetrameric enzymes with either isoform-specific or mixed subunits, and also reveals that tetramer assembly is driven by relative polypeptide abundance with no apparent preference for like subunits.

AMP Deaminase↗
PubMed2002

Characterisation of the non-peptide nociceptin receptor agonist, Ro64-6198 in Chinese hamster ovary cells expressing recombinant human nociceptin receptors.

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the opioid receptor-like receptor or nociceptin receptor (NOP). We have compared a novel non-peptide NOP agonist Ro64-6198 with N/OFQ in a series of GTPgamma35S binding and inhibition of forskolin stimulated cAMP formation assays. GTPgamma35S binding assays were performed in membranes prepared from Chinese hamster ovary cells expressing the recombinant human NOP (CHOhNOP). cAMP inhibition studies were performed in whole CHOhNOP cells. Both Ro64-6198 and N/OFQ stimulated GTPgamma35S binding with pEC50 values(95%CL) of 7.61(0.18) and 8.58(0.21) respectively. Both Ro64-6198 and N/OFQ inhibited cAMP formation with pEC50 values of 8.45(0.9) and 9.28(028) respectively. In each assay Ro64-6198 and N/OFQ were full agonists. Ro64-6198 stimulation of GTPgamma35S binding and inhibition of cAMP formation was competitively antagonised by the NOP antagonists [Nphe1]NC(1 - 13)NH2 (10microM), J-113397 (100nM) and III-BTD (1microM) with pKB values of 7.04(0.34) and 6.29(0.10), 8.65(0.34) and 7.90(0.30) and 7.59(0.22) and 7.60(0.22) respectively. Despite the slightly reduced potency of Ro64-6198 compared with N/OFQ, by virtue of high selectivity and relative metabolic stability this molecule will be of considerable use in studies of the actions of the NOP.

Animals↗
PubMed1990

Bovine natural killer-like cell responses against cell lines expressing recombinant bovine herpesvirus type 1 glycoproteins.

The murine retrovirus shuttle vector pZipNeo SV(X)1 was used to construct plasmids encoding the three major surface glycoproteins of bovine herpesvirus-1 (BHV-1). Each plasmid was transfected into D17, a canine osteosarcoma cell line sensitive to lysis by bovine NK-like cells when infected with BHV-1. After selection in G418 sulfate, cell lines expressing the recombinant gene products were sorted by flow microfluorimetry, radioimmunoprecipitated, and analyzed by SDS-PAGE for fidelity as compared to the native viral glycoproteins. Two of the three genetically engineered cell lines (gI and gIV) could successfully serve as targets to detect bovine NK-like cytolysis. These findings support and extend a previous study from our laboratory indicating a role for BHV-1 glycoproteins in the cytolytic response by bovine NK-like cells. Additionally, this study demonstrated that individual proteins are recognized by these effector cells, and that recombinant glycoproteins can direct cytolytic activity in the absence of host cell infection-associated proteins. This is the first known report of Ag directed cytotoxicity by bovine null (non-B, non-T) cells.

Animals↗
PubMed2004

Avian adenovirus CELO recombinants expressing VP2 of infectious bursal disease virus induce protection against bursal disease in chickens.

To develop a CELO virus vector that can induce protection against infectious bursal disease, CELO viruses expressing the host-protective antigen VP2 of infectious bursal disease virus (IBDV) were constructed. In the engineered recombinants, the VP2 gene (the 441-first codons of the IBDA polyprotein) was placed under the control of the CMV promoter. Two positions in the CELO genome were chosen to insert the VP2 expression cassette. The recombinants were found apathogenic, when inoculated by different routes and even at high doses (up to 10(8) per animal). Chickens vaccinated oro-nasally with these different recombinants and challenged with very virulent IBDV were found to be poorly protected. In contrast, when inoculated with one or two (subcutaneous or intradermic) injections of CELOa-VP2, the chickens showed no clinical signs and no mortality after challenge. In the vaccinated chickens, the titers of neutralization antibody reached 7-9 values, showing that protection could be explained by the induction of a sufficient humoral response. After challenge, the weight ratio Bursa of Fabricius/body was about 2.5 per thousand, a value similar to that obtained with the commercial Bur706 vaccine. However, histological lesions in the Bursa of Fabricius were observed, showing that a complete protection was not totally achieved. Contact transmission was evidenced. Protection was also obtained when inoculation of CELOa-VP2 was carried out in ovo. Prime-boost strategies were also tested with the CELOa-VP2 vector used in association with the purified VP2 antigen, or DNA encoding VP2 or a CELO vector expressing chicken myeloid growth factor (cMGF). None of these regimens were shown to substantially increase the level of protection when compared to double CELOa-VP2 inoculations. These results indicate that CELO-based vectors are useful to safely induce a strong protective immunity against vvIBDV in chickens.

Animals↗
PubMed2000

Recombinant expression and purification of smad proteins.

Smads transduce intracellular signals initiated by members of the transforming growth factor beta (TGF beta) family, including activins, TGF betas, and bone morphogenetic proteins. Recently, various models concerning the mechanism of Smad action have been proposed; however, these models are basically qualitative. Quantitative verification of the validity of the models requires significant amounts of purified Smad proteins, but purification of full-length Smad protein has not been straightforward even using recombinant protein expression systems. Here, we report purification of Smad proteins expressed in E. coli as glutathione S-transferase-fused proteins. By glutathione-Sepharose affinity purification, ATP treatment, DEAE-Sepharose and hydroxylapatite columns, expressed Smads were purified to near homogeneity as judged by SDS-PAGE; protein recovery was ca. 1 mg/l culture for Smad2 and 100 microg/l culture for Smad4. The purified Smad proteins had three known in vitro activities: Smad2 phosphorylation by TGF beta receptor complexes immunoprecipitated from COS7 cells, Smad4 binding to Smad-binding DNA element, and Smad2 interaction with calmodulin. The data suggest that purified proteins could be useful for biochemical analyses to evaluate the current models quantitatively.

Animals↗
PubMed2000

Attachment and penetration properties of bovine herpesvirus 1 recombinants expressing pseudorabies virus glycoproteins gC and gB.

OBJECTIVE: Bovine herpesvirus 1 (BHV-1) has a relatively narrow host cell range in vitro and in vivo when compared to pseudorabies virus (PrV). The aim of this study was to elucidate whether homologous glycoproteins gC and gB from PrV can function in a heterologous BHV-1 background and whether the expression of these PrV glycoproteins influences the in vitro host cell specificity of BHV-1. METHODS: We constructed BHV-1 recombinants in which PrV gC and gB were expressed either individually or in combination, and examined their attachment and penetration properties in permissive Madin-Darby bovine kidney (MDBK), semipermissive hamster lung (HmLu-1) and nonpermissive murine embryo fibroblast (A31) cells. RESULTS: Two BHV-1 recombinants which expressed PrV gC exhibited remarkable competence in virus attachment to cells. The expression of PrV gB improved the virus attachment only a little but penetration, especially into HmLu-1 and A31 cells, was enhanced. CONCLUSIONS: These results indicate that PrV gC and gB can function in a BHV-1 environment and facilitate virus attachment and penetration by BHV-1.

Animals↗
PubMed1998

[Human bone morphogenetic protein-2: recombinant expression in E. coli].

OBJECTIVE: To explore the way of producing human bone morphogenetic protein-2(hBMP-2) for bone healing by using the gene engeneering techniques. METHODS: hBMP-2 cDNA fragment, which consists of 3' end partial propeptide and mature peptide sequence, was inserted into the multiple cloning site of expression vector pkpL-3a via ligation. The recombinant plasmid pkpL-3a/hBMP-2 was transformed into E. coli pOp2136. By the method of restriction map, the positive expression clone was selected. SDS-PAGE analysis showed a new foreign protein band near 27,000 after induction. The yield of induced hBMP-2 accouned for 10% of the total bacterial proteins. RESULTS: The partial purified recombinant hBMP-2 was implanted into Wistar rat thigh. After 4 weeks, histological analysis showed that it induced the proliferation of mesenchymal type cells and formation of new cartilage and bone in the implantation area. CONCLUSION: The hBMP-2 produced by gene engeneering techniques has the biologic capacity of ectopic bone formation.

Animals↗
PubMed2005

Recombinant expression, purification, and functional analysis of two novel cystatins from sugarcane (Saccharum officinarum).

Phytocystatins are cysteine proteinase inhibitors from plants implicated in the endogenous regulation of protein turnover, programmed cell death, and in defense mechanisms against pathogens. To date, only few cystatin genes have been characterized in most plant species. We have previously characterized the protein Canecystatin, the first cystatin described in sugarcane. In an attempt to study novel Canecystatins, we identified two ORFs encoding cystatins (referred as CaneCPI-2 and CaneCPI-3) using the data from the Sugarcane EST genome project. These ORFs were then subcloned and expressed in Escherichia coli using pET28 expression vector. High amounts (approximately 20 mg/L) of pure recombinant proteins were obtained by affinity chromatography in a single step of purification. Polyclonal antibodies against the recombinant Canecystatins were raised, allowing the immunodetection of the endogenous proteins in the plant tissues. Moreover, the proteins were able to inhibit papain in a fluorometric assay with K(i) values of 0.2 and 0.25 microM for CaneCPI-2 and CaneCPI-3, respectively. These findings contribute to a better understanding of the activity of sugarcane cystatins and encourage future activity and structural studies of these proteins.

Chromatography, Affinity↗
PubMed1990

Recombinant expression, biochemical characterization, and biological activities of the human MGSA/gro protein.

Melanoma growth stimulatory activity (MGSA) is a mitogenic protein secreted by Hs294T melanoma cells that corresponds to the polypeptide encoded by the human gro gene. The MGSA/gro cDNA has been expressed in mammalian cells and the secreted recombinant factor has been purified. Biochemical and biological characterization shows that the recombinant protein is identical with the natural protein and is devoid of posttranslational glycosylation, sulfation, and phosphorylation. The two C-terminal amino acids are proteolytically removed from the mature recombinant MGSA, indicating a length of 71 instead of the predicted 73 amino acids. The recombinant MGSA is mitogenically active on the Hs294T melanoma cells. The purified MGSA competes with interleukin 8 for binding to neutrophil receptors and exhibits neutrophil chemotactic activity equivalent to that of interleukin 8.

Amino Acid Sequence↗
PubMed1991

A novel strategy for production of a highly expressed recombinant protein in an active form.

Under standard growth conditions, E. coli transformed with the high-level expression vector pMON5525 produces recombinant DMAPP/AMP transferase in inactive, insoluble complexes. We have produced large amounts of active, soluble protein by growing and inducing the cells under osmotic stress in the presence of sorbitol and glycyl betaine. This caused an increase of up to 427-fold in the active yield, and the disappearance of the protein from the pelletable fraction of cell extracts. This treatment may have wide applicability.

Alkyl and Aryl Transferases↗
PubMed1992

Nonreplicating vaccinia vector efficiently expresses recombinant genes.

Modified vaccinia Ankara (MVA), a highly attenuated vaccinia virus strain that has been safety tested in humans, was evaluated for use as an expression vector. MVA has multiple genomic deletions and is severely host cell restricted: it grows well in avian cells but is unable to multiply in human and most other mammalian cells tested. Nevertheless, we found that replication of viral DNA appeared normal and that both early and late viral proteins were synthesized in human cells. Proteolytic processing of viral structural proteins was inhibited, however, and only immature virus particles were detected by electron microscopy. We constructed an insertion plasmid with the Escherichia coli lacZ gene under the control of the vaccinia virus late promoter P11, flanked by sequences of MVA DNA, to allow homologous recombination at the site of a naturally occurring 3500-base-pair deletion within the MVA genome. MVA recombinants were isolated and propagated in permissive avian cells and shown to express the enzyme beta-galactosidase upon infection of nonpermissive human cells. The amount of enzyme made was similar to that produced by a recombinant of vaccinia virus strain Western Reserve, which also had the lacZ gene under control of the P11 promoter, but multiplied to high titers. Since recombinant gene expression is unimpaired in nonpermissive human cells, MVA may serve as a highly efficient and exceptionally safe vector.

Animals↗
PubMed2004

A carboxypeptidase inhibitor from the tick Rhipicephalus bursa: isolation, cDNA cloning, recombinant expression, and characterization.

A novel proteinaceous metallo-carboxypeptidase inhibitor, named tick carboxypeptidase inhibitor (TCI), was isolated from the ixodid tick Rhipicephalus bursa and N-terminally sequenced. The complete cDNA encoding this protein was cloned from tick mRNA by reverse transcription-PCR and rapid amplification of cDNA ends techniques. The full-length TCI cDNA contains an open reading frame coding for a precursor protein of 97 amino acid residues that consists of a predicted signal peptide of 22 residues and of mature TCI, a 75-residue cysteine-rich protein (12 Cys). The deduced amino acid sequence shows no homology to other known proteins; the C terminus, however, resembles those of other protein metallo-carboxypeptidase inhibitors, suggesting a common mechanism of inhibition. Recombinant TCI expressed in Escherichia coli is fully functional and inhibits carboxypeptidases of the A/B subfamily with equilibrium dissociation constants in the nanomolar range. Structural analyses by circular dichroism and nuclear magnetic resonance indicate that TCI is a protein strongly constrained by disulfide bonds, unusually stable over a wide pH range and highly resistant to denaturing conditions. As a tight binding inhibitor of plasma carboxypeptidase B, also known as thrombin-activatable fibrinolysis inhibitor, recombinant TCI stimulates fibrinolysis in vitro and thus may have potential for applications to prevent or treat thrombotic disorders.

Amino Acid Sequence↗
PubMed2000

Mouse cytomegalovirus in developing brain tissue: analysis of 11 species with GFP-expressing recombinant virus.

Cytomegaloviruses (CMVs) are species-specific large double-stranded DNA viruses. Mouse and human CMVs have a similar morphology, similar gene sequence, and exert similar cellular effects, but the replication of the virus outside its primary host species is limited. This may confer upon CMV certain advantages for expression of foreign genes or cellular labels in brain cells of nonhost species. We examined the ability of recombinant mouse (m)CMV expressing green fluorescent protein (GFP) to serve as a vector for transgene expression in developing neurons and glia outside the normal host species. For comparative purposes, 11 species were examined. Mouse CMV reporter gene expression was particularly strong in the developing brain of its normal host species, mouse, where it replicated in cultures and brain slices, leading to cell death. All mammalian species tested (human, rat, gerbil, hamster, mouse) showed reporter gene expression after mCMV infection. High levels of mCMV infection were also found in chicken central nervous system cells in vitro, and a low level of mCMV expression was found after an initial delay in turtle neurons and glia. No mCMV reporter gene expression was found in frog cells or aplysia neurons or glia or in drosophila or fungal cells. Infection of nonmouse neurons by low concentrations of mCMV led to strong expression of GFP in dendrites and axons with normal morphology. Despite the lack of replication, high doses of mCMV induced morphologic changes in neurons and glia from hamster and rat brain slices, leading to cells rounding up, and to the formation of giant cells consisting of an aggregate of many cells fused together into a syncytium. In contrast, in human hippocampal slices, GFP-expressing cells infected with mCMV had a relatively normal appearance 12 days after inoculation. To determine whether a CMV from another species could serve as a vector for gene transfer, a recombinant human CMV-expressing GFP was used for transgene expression in rat brain cells in vitro. Cytomegaloviruses thus have potential as useful vectors for gene transfer and labeling central nervous system cells, with the actions of CMV being dependent on a number of factors.

Animals↗