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PubMed2003

Recombinant expression of the larval excretory-secretory antigen TES-120 of Toxocara canis in the methylotrophic yeast Pichia pastoris.

A gene encoding the larval excretory-secretory antigen TES-120 of the dog ascarid worm Toxocara canis was cloned into the methylotrophic yeast Pichia pastoris. Specificity of the recombinant TES-120 antigen produced by the yeast was investigated. Forty-five human serum samples from patients infected with different()parasitic organisms, including 8 cases of toxocariasis, were tested against the recombinant antigen in immunoblot assays. Results from the assays showed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only. This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.

Animals↗
PubMed2000

Single channel analysis of a novel NMDA channel from Xenopus oocytes expressing recombinant NR1a, NR2A and NR2D subunits.

Two types of subunit (an NR1 subunit and one of the NR2 subunits) are sufficient to form efficient NMDA receptors. In order to investigate whether functional receptors may contain more than one sort of NR2 subunit, NR1, NR2A and NR2D subunits were co-expressed in Xenopus oocytes. Single channel recordings from the oocytes showed, as expected, channel openings that are characteristic of NR1/NR2A receptors (ca 50 and 40 pS conductances), and channels characteristic of NR1/NR2D receptors (ca 40 and 20 pS conductances). However, they also contained a novel channel that contained conductances of ca 30, 40 and 50 pS, with direct transitions between all three levels. It is postulated that the novel channel contains NR1, NR2A and NR2D subunits. The implications of this for receptor stoichiometry are discussed. The novel channel was intermediate between the NR1/NR2A and NR1/NR2D 'duplet' receptors in the length of the channel activations, and in the probability of being open during activation. Its glycine sensitivity was much higher than that of NR1/NR2A, and was comparable with that of the other 'duplet' receptors.

Animals↗
PubMed1995

Recombinant expression of hepatitis A virus protein 3A: interaction with membranes.

The function of hepatitis A virus (HAV) protein 3A and its structural requirements were studied in vitro and in a bacterial expression system by comparing the polypeptide precursor 3AB derived from a cytopathogenic strain with that of an attenuated strain. Although the precursor polypeptides 3AB of both HAV strains bind to microsomal membranes after translation in vitro they differ in inducing membrane permeability when expression is induced in bacteria. Intake and release of macromolecules was dramatically increased when 3AB of the cytopathogenic strain was expressed. Amino acid sequence alignments suggest that membrane binding might be due to a hydrophobic stretch near the C-terminus of 3A found in all picornaviruses whereas the ability to induce permeability of E. coli membranes is determined by an amphipathic helix formed at the N-terminus of 3A of HAVFG.

Amino Acid Sequence↗
PubMed2000

An unusual member of the human growth hormone/placental lactogen (GH/PL) family, the testicular alternative splicing variant hPL-A2: recombinant expression revealed a membrane-associated growth factor molecule.

The human growth hormone/placental lactogen (GH/PL) gene cluster consists of five highly-related genes (GH-N, GH-V, PL-L, PL-A, PL-B). This evolutionarily young gene cluster codes for an array of mRNAs and proteins, such as the major 22 k forms (hGH-N/V, identical PL-A and B), 20 k and 17.5 k hGH-N and the recently described 25 k hGH-Delta4, a presumably chimeric molecule. In addition, two longer alternatively spliced, (intron D retaining) mRNAs isoforms, termed PL-A2 and GH-V2, have been described in placenta and testis. To elucidate the role of hPL-A2 in male reproduction and pregnancy, testicular PL-A2 cDNA was cloned in a complementary overlapping 2-way RT-PCR approach to analyze translation, localization and structure/function of this unusual member of the GH/PL growth factor family. Analysis of insect mRNA revealed that intron D-retaining PL-A2 cDNA was expressed without splicing in the baculovirus expression system. Thus, PL-A2 mRNA does not represent a nuclear intermediate splicing product simply co-isolated with the mature RNA, but is a stable mRNA isoform generated by placental/testis-specific splicing factors. Recombinant protein was present in whole cell extracts, and no secreted protein was detected in the supernatant. Immunologically, the N-terminus of the 230 amino acid protein is similar to 22 k hPL-A/B, as determined by hPL-specific monoclonal antibodies. In contrast, the C-terminus shares a hydrophobic region presumably responsible for membrane insertion. By the use of confocal microscopy recombinant hPL-A2 was localized in the cell membrane. Thus, hPL-A2 might exert its function by modulating GH/PL actions or act as an independent growth-regulatory molecule itself and its functions in male reproduction and embryonic development remain to be investigated.

Aged↗
PubMed1988

Expressed recombinant cadherins mediate cell sorting in model systems.

Cadherins are cell-surface glycoproteins responsible for Ca2+-dependent cell-to-cell adhesion. E- or P-cadherin was transfected into L cells, which normally have little cadherin activity, and cellular aggregation of the resulting transfectants was observed to be a function of the cadherin molecule expressed. Transfected cells preferentially adhered to cells expressing the same cadherin subclass. Furthermore, in reconstituted embryonic lung tissue, E-cadherin-expressing L cells were associated with epithelial tubules expressing E-cadherin, while untransfected L cells associated with mesenchymal cells. These results provide the first direct evidence that the differential expression of cadherins can play a role in cell sorting in heterogeneous cell populations.

Agglutination↗
PubMed1994

Ligand-specificity of the rat GLP-I receptor recombinantly expressed in Chinese hamster ovary (CHO-) cells.

Glucagon-like peptide-I (GLP-I) is a potent incretin hormone and is considered as a new therapeutic tool in the treatment of diabetes mellitus. This study was designed to precisely characterize the binding behavior and activation of the recombinant GLP-I receptor against naturally occurring ligands of the glucagon/VIP/secretin peptide hormone family. CHO-cells were stably transfected with a plasmid containing a cDNA encoding for the rat GLP-I receptor. Northern blot analysis with this cDNA showed a single band of 2.7 kb in CHO cells, while in RINm5F cells, three bands of 2.7, 3.4, and 3.6 kb were specifically labelled. In receptor-binding studies 125I-GLP-I was displaced by GLP-I and weakly by PHI and oxyntomodulin but not by helodermin, helospectin I, helospectin II, secretin, VIP, and PACAP-38. Intracellular cAMP generation was stimulated by GLP-I, PHI, and oxyntomodulin. Helodermin, helospectin I, helospectin II, secretin, VIP, and PACAP-38 were not able to displace 125I-GLP-I from its receptor or to stimulate intracellular cAMP production. This data shows that the GLP-I receptor is characterized by a high ligand specificity.

Animals↗
PubMed1993

Recombinant expression of rat and human Gro proteins in Escherichia coli.

A full-length rat gro cDNA containing the signal sequence was inserted to a plasmid/phage vector pTD-lacs which had the Escherichia coli alkaline phosphatase leader sequence down-stream of the lac promoter. After removal of the gro signal sequence by site-directed mutagenesis, the vector was introduced to E. coli JM109. The cells grown in the presence of isopropyl beta-D-thiogalactopyranoside were found to contain the recombinant mature rat Gro protein in the periplasmic space. The protein was released from the cells by osmotic shock, and could be purified to homogeneity from the periplasmic fluid by a single-step procedure using reverse phase high performance liquid chromatography. By similar procedures, recombinant human Gro alpha could be obtained. In each case, about 10 mg of purified cytokine were obtained from 1 litre of bacterial culture.

Animals↗
PubMed1994

Humoral and cell-mediated immunity in rabbits immunized with live non-replicating avipox recombinants expressing the HIV-1SF2 env gene.

The canarypox (CP) and fowlpox (FP) viruses, which are unable to replicate productively in non-avian species, have been utilized as live vectors carrying the HIV-1SF2 env gene with the putative immunosuppressive (IS) region complete (CPIS+ and FPIS+) or deleted (CPIS- and FPIS-). To determine if these avipox-env recombinants could be utilized to elicit a specific immune response against HIV-1, six groups of rabbits were immunized with CPIS+, CPIS-, FPIS+, FPIS- constructs or their non-engineered wild-type CPwt or FPwt counterparts. After a primary inoculation and successive boosters, env-specific humoral and cell-mediated immunity were demonstrated by ELISA, immunoblots and lymphoproliferation assays. Antibody titres and neutralization activities were higher in CP- than FP-inoculated rabbits, the CPIS+ always showing a similar immunogenic capacity to CPIS-. Evidence is also presented indicating that rabbit sera possess group-specific antibodies, which were, however, unable to cross-neutralize divergent HIV-1 strains. Although the protective capacity against HIV-1 experimental infection has not yet been determined in these animals, our results suggest that these recombinants might represent promising and safer candidate vaccines against HIV-1.

AIDS Vaccines↗
PubMed2000

Comparative study of the asparagine-linked sugar chains of human lipocalin-type prostaglandin D synthase purified from urine and amniotic fluid, and recombinantly expressed in Chinese hamster ovary cells.

Lipocalin-type prostaglandin D synthase (L-PGDS) is a highly glycosylated member of the lipocalin gene family and is secreted into various human body fluids. We comparatively analyzed the structures of asparagine-linked sugar chains of human L-PGDS produced by recombinant Chinese hamster ovary cells and naturally occurring human urine and amniotic fluid. After the sugar chains were liberated by hydrazinolysis followed by N-acetylation, they were derivatized with 2-aminobenzamide. All of the sugar chains of three L-PGDSs occur as biantennary complex-type sugar chains. Most of the sugar chains of three samples were fucosylated on the inner most N-acetylglucosamine residue. Although the sugar chains of the recombinant L-PGDS do not contain any bisecting N-acetylglucosamine residues, 58% and 34% of the fucosylated-sugar chains of amniotic fluid and urine L-PGDSs, respectively, contain bisecting N-acetylglucosamine residues. The sialic acid residues occur solely as Siaalpha2-->3Gal groups of the recombinant L-PGDS; the sialic acid residues of other L-PGDS occur as both Siaalpha2-->3Gal and Siaalpha2-->6Gal groups. Variations in L-PGDS glycosylation may prove useful as markers to further elucidate the role of L-PGDS glycoforms in different tissues.

Acetylglucosamine↗
PubMed2000

Human 11beta-hydroxysteroid dehydrogenase 1/carbonyl reductase: recombinant expression in the yeast Pichia pastoris and Escherichia coli.

Detoxification of aldehydes and ketones generally proceeds via reduction to their corresponding alcohols, which are then conjugated and eliminated. We focused our interest on 11beta-hydroxysteroid-dehydrogenase type 1 (11beta-HSD 1), a pluripotent enzyme which physiologically performs the interconversion of active and inactive glucocorticoid hormones, and which also participates in xenobiotic carbonyl compound detoxification. 11beta-HSD 1 belongs to the protein superfamily of the short-chain dehydrogenases/reductases (SDR), and has been structurally and functionally characterized. 11beta-HSD 1 is a glycosylated membrane protein which is very difficult to purify in an active state. In addition, expression levels in humans differ in a wide range. In order to facilitate biochemical and molecular studies on the significance of human 11beta-HSD 1 in detoxification processes, we have successfully performed the overexpression of recombinant human 11beta-HSD 1 in the yeast Pichia pastoris and in Escherichia coli. Recombinant 11beta-HSD 1 from E. coli was purified to homogeneity and used to generate a polyclonal antibody. The enzyme had no enzymatic activity, possibly due to the lack of glycosylation and/or incorrect folding in E. coli. In contrast, 11beta-HSD 1 overexpressed in P. pastoris was enzymatically active towards its physiological glucocorticoid substrates as well as towards xenobiotic carbonyl compounds. In western blot experiments the antibody crossreacted with both recombinant 11beta-HSD 1 forms and with the native enzyme from mouse and human liver. In conclusion, recombinant 11beta-HSD 1 from P. pastoris serves as a valuable tool for future studies on carbonyl compound detoxification.

11-beta-Hydroxysteroid Dehydrogenase Type 1↗
PubMed1991

Complete sequence of the allergen Amb alpha II. Recombinant expression and reactivity with T cells from ragweed allergic patients.

This study defines the complete primary structure of Amb alpha II, an important allergen produced by short ragweed (Ambrosia artemisiifolia). The deduced amino acid sequence derived from the cDNA indicates that Amb alpha II shares approximately 65% sequence identity with the Amb alpha I multigene family of allergens. Full-length cDNA encoding Amb alpha I.1 and Amb alpha II have been expressed in E. coli and purified. An in-frame linker encoding polyhistidine has been added to the 5' end of the cDNA to facilitate purification using Ni2+ ion affinity chromatography, yielding greater than 90% pure recombinant protein in a single step. T cells from patients allergic to ragweed proliferate in response to pollen extract as well as purified recombinant Amb alpha I.1 and Amb alpha II. T cell lines established using either Amb alpha I.1 or II as the stimulating Ag exhibit a high level of cross-reactivity to both proteins. This result is entirely consistent with the extensive primary sequence identity shared by these two proteins. These data suggest that allergic humans recognize shared T cell epitopes on these two related molecules.

Allergens↗
PubMed1997

Exclusion of bioactive contaminations in Streptococcus pyogenes erythrogenic toxin A preparations by recombinant expression in Escherichia coli.

The streptococcal erythrogenic exotoxin A (SPEA) belongs to the family of bacterial superantigens and has been implicated in the pathogenesis of a toxic shock-like syndrome and scarlet fever. Concerning its biological activity, mainly T-cell-stimulatory properties, conflicting data exist. In this study, we show that most of the SPEA preparations used so far contain biologically active contaminations. Natural SPEA from the culture supernatant of Streptococcus pyogenes NY-5 and recombinant SPEA purified from the culture filtrate of S. sanguis are strongly contaminated with DNases. We show that natural SPEA induces more tumor necrosis factor alpha (TNF-alpha) than recombinant SPEA, but we also show that DNases are able to induce TNF-alpha. In commercial SPEA preparations, we identified a highly active protease, which was shown not to be SPEB. To exclude these contaminations, we overexpressed SPEA cloned in the effective high-level expression vector pIN-III-ompA2 in Escherichia coli. The expressed SPEA shows the same amino acid composition as natural SPEA, whereas functional studies reported so far were carried out with toxins containing an incorrect amino terminus. We describe the rapid purification of lipopolysaccharide-, DNase-, and protease-free SPEA in two steps from the host's periplasm and its structural characterization by circular dichroism. Our results represent for the first time the production in E. coli of recombinant SPEA with the authentic N-terminal sequence and a proven superantigenic activity. Collectively, our results indicate that immunological studies of superantigens require highly purified substances free of biologically active contaminations.

Animals↗
PubMed2005

Molecular cloning, recombinant expression and IgE-binding epitope of omega-5 gliadin, a major allergen in wheat-dependent exercise-induced anaphylaxis.

Wheat omega-5 gliadin has been identified as a major allergen in wheat-dependent exercise-induced anaphylaxis. We have detected seven IgE-binding epitopes in primary sequence of the protein. We newly identified four additional IgE-binding epitope sequences, QQFHQQQ, QSPEQQQ, YQQYPQQ and QQPPQQ, in three patients with wheat-dependent exercise-induced anaphylaxis in this study. Diagnosis and therapy of food allergy would benefit from the availability of defined recombinant allergens. However, because omega-5 gliadin gene has not been cloned, recombinant protein is currently unavailable. We sought to clone the omega-5 gliadin gene and produce the homogeneous recombinant protein for use in an in vitro diagnostic tool. Using a PCR-based strategy we isolated two full-length omega-5 gliadin genes, designated omega-5 and omega-5b, from wheat genomic DNA and determined the nucleotide sequences. The protein encoded by omega-5a was predicted to be 439 amino acids long with a calculated mass of 53 kDa; the omega-5b gene would encode a 393 amino acid, but it contains two stop codons indicating that omega-5b is pseudogene. The C-terminal half (178 amino acids) of the omega-5a gliadin protein, including all 11 IgE-binding epitope sequences, was expressed in Escherichia coli by means of the pET system and purified using RP-HPLC. Western blot analysis and dot blot inhibition assay of recombinant and native omega-5 gliadin purified from wheat flour demonstrated that recombinant protein had IgE-binding ability. Our results suggest that the recombinant protein can be a useful tool for identifying patients with wheat-dependent exercise-induced anaphylaxis in vitro.

Allergens↗
PubMed1997

Characterization of a Pasteurella multocida plasmid and its use to express recombinant proteins in P. multocida.

The complete nucleotide sequence of a naturally occurring 5.36-kb streptomycin and sulphonamide resistance plasmid, designated pIG1, isolated from type D Pasteurella multocida was determined. A 1.6-kb noncoding region and a 1.4-kb region encoding three putative proteins were shown by sequence homologies and functional characterizations to be involved in the replication and mobilization of pIG1, respectively. The remaining sequence carried an unusual arrangement of streptomycin- and sulphonamide-resistant genes when compared to various other plasmids. It appears that the antibiotic resistance region of pIG1 may have evolved by recombination between three different short direct repeat DNA sequences. A 4.5-kb recombinant plasmid was constructed by replacing the antibiotic resistance genes of pIG1 with a kanamycin resistance gene and seven unique restriction sites. The resulting plasmid, designated pIG112, stably replicates in P. multocida, Pasteurella haemolytica, Actinobacillus pleuropneumoniae, and Escherichia coli and can be introduced into these organisms by either transformation or conjugation. This vector exists at approximately 70 copies per cell in P. multocida and approximately 20 copies per cell in E. coli. To demonstrate plasmid-borne gene expression in P. multocida, the P. multocida dermonecrotic toxin gene, toxA, and a genetically modified form of this gene were cloned into pIG112 and expressed in high amounts in a nontoxigenic P. multocida strain. Cell culture assays demonstrated that nontoxigenic P. multocida expressing toxA was cytopathic, whereas a strain expressing the modified toxA derivative was not.

Animals↗
PubMed1985

Vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D prevents latent herpes in mice.

In humans, herpes simplex virus causes a primary infection and then often a latent ganglionic infection that persists for life. Because these latent infections can recur periodically, vaccines are needed that can protect against both primary and latent herpes simplex infections. Infectious vaccinia virus recombinants that contain the herpes simplex virus type 1 (HSV-1) glycoprotein D gene under control of defined early or late vaccinia virus promoters were constructed. Tissue culture cells infected with these recombinant viruses synthesized a glycosylated protein that had the same mass (60,000 daltons) as the glycoprotein D produced by HSV-1. Immunization of mice with one of these recombinant viruses by intradermal, subcutaneous, or intraperitoneal routes resulted in the production of antibodies that neutralized HSV-1 and protected the mice against subsequent lethal challenge with HSV-1 or HSV-2. Immunization with the recombinant virus also protected the majority of the mice against the development of a latent HSV-1 infection of the trigeminal ganglia. This is the first demonstration that a genetically engineered vaccine can prevent the development of latency.

Animals↗
PubMed2005

p73 beta-expressing recombinant adenovirus: a potential anticancer agent.

Tumor suppressor p53-based gene therapy strategy is ineffective in certain conditions. p73, a p53 homologue, could be a potential alternative gene therapy agent as it has been found to be an important determinant of chemosensitivity in cancer cells. Previously, we have reported the generation of a replication-deficient adenovirus expressing p73 beta (Ad-p73). In this study, we evaluated the therapeutic potential of Ad-p73 against a panel of cancer cells (n=12) of different tissue origin. Ad-p73 infected all the cell lines tested very efficiently resulting in several-fold increase in p73 beta levels, which is also functional as it activated the known target gene p21(WAF1/CIP1). Infection with Ad-p73 resulted in potent cytotoxicity in all the cell lines tested. The mechanism of p73-induced cytotoxicity in these cell lines is found to be due to a combination of cell cycle arrest and induction of apoptosis. In addition, exogenous overexpression of p73 by Ad-p73 infection increased the chemosensitivity of cancer cells by many fold to commonly used drug adriamycin. Moreover, Ad-p73 is more efficient than Ad-p53 in enhancing the chemosensitivity of mutant p53 harboring cells. Furthermore, Ad-p73 infection did not induce apoptosis in human normal lung fibroblasts (HEL 299) and human immortalized keratinocytes (HaCaT). These results suggest that Ad-p73 is a potent cytotoxic agent specifically against cancer cells and could be developed as a cancer gene therapy agent either alone or in combination with chemotherapeutic agents.

Adenoviridae↗
PubMed2005

Ca2+ signaling in HEK-293 and skeletal muscle cells expressing recombinant ryanodine receptors harboring malignant hyperthermia and central core disease mutations.

Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca(2+) release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca(2+) pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca(2+) homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca(2+) revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca(2+) transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca(2+) derangements of different degrees account for the specific cellular phenotypes of the two disorders.

Aequorin↗