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At least 397 records · Page 22Linked to original sources
PubMed2000

Recombinant expression analysis of natural and synthetic bovine alpha-casein in Escherichia coli.

As a prelude to developing a yeast-based fermentation process for the production of phenylalanine-free alpha-casein as a foodstuff for patients suffering from phenylketonuria, we cloned the gene encoding bovine alpha-casein. We synthesised a modified gene sequence encoding the same, but devoid of phenylalanine codons and with a codon bias similar to that of naturally occurring highly expressed genes in Saccharomyces cerevisiae. The results show that both gene sequences are readily expressed in Escherichia coli when cloned in an E. coli bacteriophage T7 promoter-driven plasmid vector. In this host, the natural and synthetic casein proteins were produced at levels equating to 18.0% and 7.6% of the cell's soluble protein, respectively.

Amino Acid Sequence↗
PubMed1997

Cloning, sequencing, and recombinant expression of the porcine inhibitor of carbonic anhydrase: a novel member of the transferrin family.

The plasma from many vertebrates contains a component that specifically binds and inhibits carbonic anhydrase II with nanomolar affinity. Amino-terminal sequencing of pICA, the previously identified 79-kDa carbonic anhydrase inhibitor isolated from porcine plasma [Roush, E. D., & Fierke, C. A. (1992) Biochemistry 31, 12536-12542], and sequencing of four proteolytic fragments of pICA revealed that each of the partial sequences has 40-80% sequence identity with members of the transferrin protein family. We describe here the isolation of a full-length cDNA clone of pICA from a lambda gt11 porcine liver cDNA library. Heterologous expression of this cDNA clone in a Pichia pastoris expression system led to the secretion into the medium of 5 mg/L of a 79-kDa protein that specifically reacts with anti-pICA antibodies and binds tightly to a carbonic anhydrase-Sepharose affinity column. Pairwise sequential alignment of pICA with various transferrins reveals an amino acid identity as high as 64% and predicts that 16 transferrin disulfide bonds are conserved. However, despite these structural similarities, the properties of pICA are distinct from the properties of transferrin. pICA exhibits a significantly decreased affinity for iron that can be attributed to the loss of one of the eight amino acids that coordinate iron in the transferrins as well as both of the arginine residues responsible for anion binding. In addition, the antigenic determinants of pICA and the transferrins are not identical. These data imply that pICA, along with saxiphilin, is a member of a diverse superfamily of transferrin-like proteins with functions other than iron binding.

Amino Acid Sequence↗
PubMed2005

Primary structure, recombinant expression, and molecular characterization of Phl p 4, a major allergen of timothy grass (Phleum pratense).

Grass pollen allergy is one of the most important allergic diseases world-wide. Several meadow grasses, like timothy grass and rye grass, contribute to allergic sensitizations, but also allergens from extensively cultivated cereals, especially rye, make a profound contribution. The group 4 allergens are well known as important major allergens of grasses. We have cloned for the first time group 4 sequences from Phleum pratense, Lolium perenne, Secale cereale, Triticum aestivum, and Hordeum vulgare, and investigated the IgE-reactivity of recombinant Phl p 4 as a candidate for allergy diagnostic and therapeutic applications.

Allergens↗
PubMed2003

Recombinant expression of Toxocara canis excretory-secretory antigen TES-120 in Escherichia coli.

The gene encoding the excretory-secretory antigen TES-120 of dog ascarid worm Toxocara canis was cloned into the bacterium Escherichia coli. The specificity of the recombinant TES-120 antigen produced by the bacterium was investigated. A total of 45 human serum samples from patients infected with differenthelminthes and protozoa, including 8 cases of toxocariasis, were tested against the recombinant antigens in immunoblot assays. The results from the assays revealed 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↗
PubMed2006

[Construction of eukaryotic expression recombinant plasmid of trichomonas vaginalis ferredoxin gene].

Total DNA was extracted from T. vaginalis with Chelex-100 method and used as templates for PCR. The ferredoxin gene was directionally cloned into plasmid pMD-18T vector and subcloned into eukaryotic expression vector pcDNA3. 1 (+). The transformants were screened and identified by PCR and restriction analysis. The size of amplified ferredoxin gene was 306bp and the DNA sequence of cloned gene was same with that published.

Animals↗
PubMed1992

Novel cell lines derived from transgenic mice expressing recombinant human proteins. Transgenic hepatoma-derived cell lines.

We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human alpha 1-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of an onc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human alpha 1-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing.

Animals↗
PubMed1994

Recombinant expression and structural and binding properties of alpha 1(VI) and alpha 2(VI) chains of human collagen type VI.

Full-length alpha 1(VI) and alpha 2(VI) cDNAs in an eukaryotic expression vector were used to obtain stably transfected human kidney cell clones and to purify these collagen-VI chains in substantial quantities from the culture medium. Both chains appeared mainly as monomers together with some dimers that were disulfide linked through their C-terminal globular domains. Despite sufficient hydroxylation of proline and lysine residues, the chains did not form a triple-helix, as shown by electronmicroscopy, CD spectra and pepsin sensitivity. Digestion of the chains with bacterial collagenase released the N-terminal and C-terminal globular domains, which were identified by their size and partial sequences. They showed a substantial content of alpha-helical conformation and a distinct globular structure after rotary shadowing. Antibodies could be raised that distinguished between the two chains and reacted with the globular domains. The alpha 2(VI) but not the alpha 1(VI) chain showed binding to a heparan sulfate proteoglycan (perlecan), fibronectin and pepsin-solubilized collagen VI. Purified globular domains did not bind these ligands indicating the localization of binding sites within the triple-helical domain. Both chains showed a distinct affinity for heparin but failed to bind to various collagen types.

Amino Acid Sequence↗
PubMed1994

Cloning and recombinant expression of a novel human low molecular weight Ca(2+)-dependent phospholipase A2.

Extensive biochemical studies of phospholipase A2s (PLA2s) over the last two decades indicate that there are likely to be several distinct PLA2 genes in mammals. Here we report the cloning of a 1-kilobase pair cDNA encoding a novel human low molecular weight PLA2. The cDNA appears to encode a 118-amino acid mature peptide (M(r) = 13,592) preceded by a 20-residue prepeptide. The deduced amino acid sequence encodes a protein that lacks one of the seven disulfide bridges found in similar PLA2s and, therefore, represents a class of enzymes distinct from the mammalian group I and group II enzymes. An RNA blot hybridized with the cDNA exhibited a putative 1.2-kilobase pair transcript in heart and, less abundantly, in lung, as well as multiple putative transcripts in placenta. When the cDNA was expressed using an Epstein-Barr virus-based vector in human 293s cells, PLA2 activity accumulated in the culture medium. Conditioned medium optimally hydrolyzed the phospholipids of [1-14C]oleate-labeled Escherichia coli at neutral to alkaline pH with 10 mM or greater Ca2+. In assays done with individual substrates, L-alpha-1-palmitoyl-2-oleoyl phosphatidylcholine was more efficiently hydrolyzed than L-alpha-1-palmitoyl-2-arachidonyl phosphatidylcholine, L-alpha-1-palmitoyl-2-arachidonyl phosphatidylethanolamine, or L-alpha-1-stearoyl-2-arachidonyl phosphatidylinositol.

Amino Acid Sequence↗
PubMed1996

An adenovirus recombinant expressing the spike glycoprotein of porcine respiratory coronavirus is immunogenic in swine.

The full-length spike (S) gene of porcine respiratory coronavirus (PRCV) was inserted into the genome of human adenovirus type 5 downstream of the early transcription region 3 promoter. The recombinant virus replicated in cultures of the swine testicle ST cell line and directed the synthesis of S antigen with a maximum yield of approximately 26 micrograms per 10(6) cells. The antigen was cell-associated except in the late phase of the infection, when a small amount (3.5 micrograms per 10(6) cells) was released. The cell-associated antigen consisted of polypeptides of molecular mass 160 kDa and 175 kDa, comigrating with the authentic precursor S' and the mature S protein of PRCV, respectively. The extracellular recombinant antigen corresponded to the 175 kDa mature protein. Some recombinant S protein was exposed on the cell surface and was recognized by neutralization-mediating anti-S monoclonal antibodies. Piglets, inoculated oronasally with the recombinant adenovirus vector developed PRCV-neutralizing serum antibodies and were partially protected against PRCV challenge, demonstrating the potential of live adenovirus as vaccine vector.

Adenoviruses, Human↗
PubMed1996

Cell-specific signal transduction of parathyroid hormone (PTH)-related protein through stably expressed recombinant PTH/PTHrP receptors in vascular smooth muscle cells.

PTH-related protein activates a G protein-coupled PTH/PTHrP receptor in many cell types and produces diverse biological actions. To study the signal transduction events associated with biological activity of the PTH/PTHrP receptor in vascular smooth muscle, a principal PTHrP-responsive tissue, rat aortic smooth muscle cells (A10) were stably transfected with a plasmid encoding a PTH/PTHrP receptor and tested for ligand binding, PTHrP-(1-34)-induced cAMP levels, inositol phosphate production, and cytosolic calcium transients. Of nineteen G418-resistant lines recovered, all exhibited high affinity binding [approximately dissociation constant (Kd) > 10(-10)) of iodinated [Tyr36]hPTHrP(1-36)NH2 and ligand-induced cAMP accumulation (2- to 100-fold), which was directly proportional to PTH/PTHrP receptor number (range 4 x 10(3) to 7 x 10(7) sites/cell]. PTHrP had no effect on intracellular calcium or inositol phosphate formation in any cell line regardless of receptor number despite the presence of detectable G alpha q). Transient overexpression of individual G alpha q proteins (G alpha q, G alpha 11 or G alpha 14) into PTH/PTHrP receptor-expressing A10 cells conferred the ability of PTHrP to increase intracellular calcium and inositol phosphate formation. Ligand activation of the recombinant PTH/PTHrP receptor elicited appropriate downstream biological effects in A10 cells including inhibition of DNA synthesis and osteopontin messenger RNA (mRNA) expression. Thus, a single PTH/PTHrP receptor, though capable of coupling to different G proteins, signals exclusively through a cAMP-dependent pathway in vascular smooth muscle.

Animals↗
PubMed1996

Cytotoxic effects of kainate ligands on HEK cell lines expressing recombinant kainate receptors.

Exposure of neurons either for prolonged periods of time or to high concentrations of excitatory amino acids (EAA), such as glutamate, results in neuronal death. Kainate also causes cell toxicity through the glutamate receptors. However, it is unclear whether the kainate receptor itself mediates any of the toxic responses. In the present study, HEK cells expressing the GluR6 +/- KA2 receptor subunit(s) were studied for their susceptibility to toxicity through the kainate receptor by kainate ligands. The natural ligand, glutamate, did not result in toxicity to the recombinant cell lines over that observed with the untransfected HEK cells, whereas kainate produced a 2-3-fold increase in LDH in both the HEK/GluR6 (ANOVA, P = 0.0001) and HEK/GluR6 + KA2 (ANOVA, P = 0.0002) cell lines following treatment with various dosages, but did not affect the HEK cells. Similar 2-3-fold increases in LDH activity were detected in both recombinant cell lines following treatment with 100 nM of SYM2081 ((2S,4R)-4-methylglutamic acid), a dose at which agonistic activity is elicited. The rank order potencies for eliciting toxicity are consistent with the previously reported EC50 values (SYM2081 > kainate > > > glutamate). Surprisingly, the kainate antagonist, NBQX, was the most toxic of the compounds tested although it had an affinity for the kainate receptor similar to glutamate. Treatment with as little as 10 nM elicited a dramatic increase in toxicity (6-10-fold) in the recombinant cell lines. At 1 microM, NBQX was significantly more toxic (Fisher PLSD, P < 0.05) than any of the other compounds tested. Thus, it appears that cell toxicity can be mediated via kainate receptor through two independent mechanisms: activation and blockage of the kainate receptor.

Cell Line↗
PubMed2005

Immune responses of mice to vaccinia virus recombinants expressing either Listeria monocytogenes partial listeriolysin or Brucella abortus ribosomal L7/L12 protein.

The Brucella abortus L7/L12 gene encoding ribosomal protein L7/L12 and the Listeria monocytogenes partial hly gene encoding the protective region of the hemolysin (partial listeriolysin, pLLO) were cloned into vaccinia virus by homologous recombination to produce recombinants WRL7/L12 and WRpLLO, respectively. The ability of these recombinants to induce humoral, cell mediated and protective immune response in mice was assessed. Although mice inoculated with WRL7/L12 recombinant produced antibodies specific to vaccinia virus and L7/L12 antigens, they were not protected against a virulent challenge with B. abortus 2308 strain. In contrast, mice inoculated with WRpLLO were protected against a challenge with virulent L. monocytogenes. Stimulation with purified fusion listeriolysin protein (MBP-LLO), but not with unrelated control protein (MBP), induced splenocytes from WRpLLO-inoculated mice to secrete significantly higher amounts of IFN-gamma than saline inoculated mice. Mice inoculated with either WRpLLO or WRL7/L12 recombinants produced predominantly IgG2a isotype antibody responses, indicative of a Th1 type of immune response. The protective potential of the WRpLLO recombinant correlated with the level of IFN-gamma produced in these mice.

Animals↗
PubMed1998

Fowl adenovirus recombinant expressing VP2 of infectious bursal disease virus induces protective immunity against bursal disease.

The right hand end Nde I fragment 3 (90.8-100 map units) of the fowl adenovirus serotype 10 (FAV-10) was characterised so as to allow the location of an insertion site for recombinant vector construction. Infectious bursal disease virus (IBDV) VP2 gene from the Australian classical strain 002/73, under the control of the FAV-10 major late promoter/leader sequence (MLP/LS) was inserted into a unique Not I site that was generated at 99.5 map units. This recombinant virus was produced without deletion of any portion of the FAV-10 genome. When administered to specific pathogen free (SPF) chickens intravenously, intraperitoneally, subcutaneously or intramuscularly, it was shown that the FAV-10/VP2 recombinant induced a serum VP2 antibody response and protected chickens against challenge with IBDV V877, an intermediate virulent classical strain. Birds were not protected when the recombinant was delivered via the conjunctival sac.

Animals↗
PubMed1995

Recombinant expression of the TGEV membrane glycoprotein M.

We have previously shown that the membrane protein M of TGEV is involved in efficient induction of alpha interferon (IFN alpha) synthesis by non-immune peripheral blood mononuclear cells incubated with fixed, TGEV-infected cells or inactivated virions. In order to determine whether M protein is able to induce interferon in the absence of other viral factors, we expressed the protein either stably in the porcine ST cells or transiently in the simian COS7 cells. Although showing no obvious difference in intracellular localization or glycosylation compared to its viral counterpart, the recombinant molecule failed to induce significant IFN activity.

Animals↗
PubMed1994

Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge.

The nucleoprotein (NP) of influenza A virus is the dominant antigen recognized by influenza virus-specific cytotoxic T lymphocytes (CTLs), and adoptive transfer of NP-specific CTLs protects mice from influenza A virus infection. BALB/c mouse cells (H-2d) recognize a single Kd-restricted CTL epitope of NP consisting of amino acids 147 to 155. In the present study, mice were immunized with various vaccinia virus recombinant viruses to examine the effect of the induction of primary pulmonary CTLs on resistance to challenge with influenza A/Puerto Rico/8/34 virus. The minigene ESNP(147-155)-VAC construct, composed of a signal sequence from the adenovirus E3/19K glycoprotein (designated ES) and expressing the 9-amino-acid NP natural determinant (amino acids 147 to 155) preceded by an alanine residue, a similar minigene NP(Met 147-155)-VAC lacking ES, and a full-length NP-VAC recombinant of influenza virus were analyzed. The two minigene NP-VAC recombinants induced a greater primary pulmonary CTL response than the full-length NP-VAC recombinant. However, NP-specific CTLs induced by immunization with ESNP(147-155)-VAC did not decrease peak virus titer or accelerate clearance of virus in the lungs of mice challenged intranasally with A/PR/8/34. Furthermore, NP-specific CTLs induced by immunization did not protect mice challenged intranasally with a lethal dose of A/PR/8/34. Sequence analysis of the NP CTL epitope of A/PR/8/34 challenge virus obtained from lungs after 8 days of replication in ESNP(147-155)-VAC-immunized mice showed identity with that of the input virus, demonstrating that an escape mutant had not emerged during replication in vivo. Thus, in contrast to adoptively transferred CTLs, pulmonary NP-specific CTLs induced by recombinant vaccinia virus immunization do not have protective in vivo antiviral activity against influenza virus infection.

Amino Acid Sequence↗
PubMed2006

[Recombinant expression of mutant human annexin V].

OBJECTIVE: To mutate human annexin V gene and transform it to Pichia Pastoris for mutant human annexin V expression, so as to be purified as active annexin V with endogenous metal chelating site. METHODS: The 5' and 3' end of native annexin V gene were mutated by specific primers. The mutant annexin V gene was inserted into pPIC9K and sequenced. The correct plasmid was linearized and transformed into Pichia Pastoris strain GS115 by electroporation. The transformants were selected from MD plates and cultured in BMGY medium and induced with methanol. The culture was centrifuged and the supernatant was analyzed by SDS-PAGE and silver staining. The binding activity of mutant human annexin V from culture supernatant was determined with phosphatidylserine exposed erythrocytes and fluorescein isothiocyanate-annexin V. RESULTS: The 5' end of native human annexin V gene was fused with GCAGGCGGCTGCGGCCAT coding sequence and 3' end 946-948 site TGT was mutated to AGC. Pichia Pastoris transformants secreted proteins of relative molecular mass 36 000 48 h after methanol induction. The concentration of this protein that inhibited 50% of the binding of fluorescein-annexin V was 4nmol/L. CONCLUSION: Highly-active recombinant mutant human annexin V with endogenous metal-chelating sites can be expressed in Pichia Pastoris system.

Annexin A5↗