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PubMed1998

[Expression, purification and characterization of recombinant human cyclophilin A].

Plasmid-derived expression of the human CyPA in E. coli would make it possible to obtain ample protein quantities and to avoid difficult task of obtaining human tissues for protein purification. The cDNA encoding human CyPA from MT4 lymph cell line has been cloned and an expression vector (pET11/CyPA) has been constructed under control of the T7 promoter for efficient expression in E. coli. The recombinant CyPA is produced at 41% of total soluble cell protein, and showed the peptidyl-prodyl cis-trans isomerase activity.

Cell Line↗

PubMed1989

Characterization of recombinant thioesterase and acyl carrier protein domains of chicken fatty acid synthase expressed in Escherichia coli.

Fatty acid synthase of animal tissue is a multifunctional enzyme comprised of two identical subunits, each containing seven partial activities and a site for the prosthetic group, 4'-phosphopantetheine (acyl carrier protein). We have recently isolated cDNA clones of chicken fatty acid synthase coding for the dehydratase, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase domains (Chirala, S.S., Kasturi, R., Pazirandeh, M., Stolow, D.T., Huang, W.Y., and Wakil, S.J. (1989) J. Biol. Chem. 264, 3750-3757). To gain insight into the structure and function of the various domains, the portion of the cDNA coding for the acyl carrier protein and thioesterase domains was expressed in Escherichia coli by using an expression vector that utilizes the phage lambda PL promoter. The recombinant protein was efficiently expressed and purified to near homogeneity using anion-exchange and hydroxyapatite chromatography. As expected from the coding capacity of the cDNA expressed, the protein has a molecular weight of 43,000 and reacts with antithioesterase antibodies. The recombinant thioesterase was found to be enzymatically active and has the same substrate specificity and kinetic properties as the native enzyme of the multifunctional synthase. Treatment of the recombinant protein with alpha-chymotrypsin results in the cleavage of the acyl carrier protein and thioesterase domain junction sequence at exactly the same site as with native fatty acid synthase. The amino acid composition of the purified recombinant protein revealed the presence of 0.6 mol of beta-alanine/mol of protein, indicating partial pantothenylation of the recombinant acyl carrier protein domain. These results indicate that the expressed protein has a conformation similar to the native enzyme and that its folding into functionally active domains is independent of the remaining domains of the multifunctional synthase subunit. These conclusions are consistent with the proposal that the multifunctional synthase gene has evolved from fusion of component genes.

Acyl Carrier Protein↗

PubMed1997

Adenoviral transfection of isolated pancreatic islets: a study of programmed cell death (apoptosis) and islet function.

Gene therapy provides a potential technique to modify immunity in vitro and therefore may prolong graft survival in vivo. However, viral infection and gene transfer may damage target cells and interfere with biologic function. Viruses, including adenovirus, are known to be capable of modulating apoptosis and initiating cell death by either inducing or suppressing specific processes, depending on the virus and cell system studied. The effect of adenovirus on islet cell viability and function has not been examined in detail. In this study, the dose-dependent effect of an adenoviral vector on islet cell death and glucose-stimulated insulin secretion (GSIS) was investigated to establish a therapeutic window for the dose of viral vector administered. Isolated pancreatic rat islets were incubated with an adenovirus expressing a beta-galactosidase gene (AdHCMVsp1LacZ) at different viral concentrations [multiplicity of infection (MOI) 1:10, 1:100, and 1:1000]. Transfection rate, in vitro and in vivo islet viability, and occurrence of programmed cell death were determined 1, 3, and 7 days after transfection. Islets, transfected at MOI 1:10 and 1:100, demonstrated apoptosis not significantly different from nontransfected controls. Islets, transfected at MOI 1:1000, demonstrated a significant increase in apoptosis at 24 hr, which decreased over 7 days of culture. The increase in apoptosis was not reflected by a significant decrease in in vitro GSIS of surviving islet cells, as assessed by stimulation index following in vitro perifusion. SCID or nude mice transplanted with AdlacZ-transfected islets (MOI 1:100 and 1:1000) remained normoglycemic for > or = 30 days. These results demonstrate that transfection of islets using adenoviral vectors can be manipulated such that efficient expression of the gene product encoded by the transfected gene (beta-galactosidase) can be achieved at lower transfecting concentrations of the adenoviral vector (MOI 1:10, 20.2%; MOI 1:100, 30.7%) while preserving islet function. This efficiency of transfection may allow pretransplant manipulation of isolated islet cells without vector-specific alteration of islet function. In cases where high virus concentrations are required for efficient gene transfer (adequate expression of the transgene product), a deleterious effect of the vector on islet cell function, with increased cell loss due to increased apoptotic events, is predicted. Using the AdlacZ vector, cell loss by apoptotic mechanisms appears limited to the first days following coculture with high viral concentrations, and does not appear to influence in vitro or in vivo cell function of the surviving islet cells.

Adenoviridae↗

PubMed1993

Construction of an expression and site-directed mutagenesis system of haloalkane dehalogenase in Escherichia coli.

Haloalkane dehalogenase from Xanthobacter autotrophicus was efficiently expressed in Escherichia coli BL21 (DE3) and E. coli JM101. After introduction of restriction sites by PCR the haloalkane dehalogenase gene (dhlA) was translationally fused behind the T7 (phi 10), trc, and tac promoters. This resulted in expression at 30 degrees C up to 38 and 18% of the total soluble cellular protein with the T7 and trc promoters, respectively. Dehalogenase expression under control of the tac promoter was below 1% of the soluble cell protein, however. Aggregation of haloalkane dehalogenase into inclusion bodies was found during growth at 37 degrees C but not at 30 degrees C. Aggregates were also formed from intact enzyme upon incubation at 37 degrees C of cells or crude extracts containing active mature dehalogenase. The high level of expression resulted in a short purification procedure in which 30-35 mg highly enriched haloalkane dehalogenase was obtained from an 0.51 culture. For the production of single-stranded DNA an f1(+) origin was introduced in the T7 expression system.

Amino Acid Sequence↗

PubMed1993

Stimulation of gene expression by introns: conversion of an inhibitory intron to a stimulatory intron by alteration of the splice donor sequence.

Efficient expression of many mammalian genes depends on the presence of at least one intron. We previously showed that addition of almost any of the introns from the mouse thymidylate synthase (TS) gene to an intronless TS minigene led to a large increase in expression. However, addition of intron 4 led to a reduction in minigene expression. The goal of the present study was to determine why TS intron 4 was unable to stimulate expression. Insertion of intron 4 into an intron-dependent derivative of the ribosomal protein L32 gene did not lead to a significant increase in expression, suggesting that its inability to stimulate expression was due to sequences within the intron. Deleting most of the interior of intron 4, improving the putative branch point, removing purines from the pyrimidine stretch at the 3' end of the intron, or removing possible alternative splice acceptor or donor sites within the intron each had little effect on the level of expression. However, when the splice donor sequence of intron 4 was modified so that it was perfectly complementary to U1 snRNA, the modified intron 4 stimulated expression approximately 6-fold. When the splice donor site of TS intron 1 (a stimulatory intron) was changed to that of TS intron 4, the modified intron 1 was spliced very inefficiently and lost the ability to stimulate mRNA production. Our observations support the idea that introns can stimulate gene expression by a process that depends directly on the splicing reaction.

Animals↗

PubMed2001

[Expression of recombinant human Flt3 ligand in Escherichia coli and its purification and characterization].

OBJECTIVE: To establish a highly efficient expression system of recombinant human Flt3 ligand (rhFL) in E. coli and a suitable purification method of the expressed products. METHOD: Human FL encoding cDNA was introduced into pProEXHT plasmid to express a 6 x His-FL fusion protein in E. coli. The fusion protein expressed in inclusion body was isolated, solubilized and refolded, and then purified by chromatography on a metal-chelating affinity column (MCAC). Its activity was detected by stimulating the proliferation of CD(34)(+) cells. RESULT: The amount of rhFL expressed was about 15% of total bacterial proteins and the purity of rhFL was 90% after MCAC. The combination of rhFL, granulocyte colony-stimulating factor and erythropoietin could stimulate CD(34)(+) cells to a 400 fold expansion. CONCLUSION: The purified rhFL had a potent activity to stimulate hematopoietic stem cells to expanse in vitro.

Cell Proliferation↗

PubMed1983

Transcription of a cloned Xenopus laevis H4 histone gene in the homologous frog oocyte system depends on an evolutionary conserved sequence motif in the -50 region.

A cloned Xenopus laevis H4 histone gene has been expressed in the X.laevis oocyte nucleus. The homologous histone H4 gene can be correctly and efficiently expressed in the frog oocyte even in presence of bacterial vector DNA. As revealed by both analytical gel electrophoresis and S1 mapping, two H4 mRNAs are specified with different transcriptional efficiencies from the tandemly repeated promoter. Results from deletion mapping of the sequences essential for promoting H4 transcription show that drastic reduction of transcription is obtained when the sequences lying between -64 and -35 bp from the mRNA cap site are removed. We demonstrate by DNA sequence comparison using a novel computer program that this important area of the H4 promoter contains two highly conserved DNA motifs near positions -51 to -46 upstream from the cap site in all H4 gene promoters analysed.

Amino Acid Sequence↗

PubMed1991

Direct expression of Bordetella pertussis filamentous hemagglutinin in Escherichia coli and Salmonella typhimurium aroA.

Nonfused (i.e., nonhybrid) filamentous hemagglutinin (FHA) of Bordetella pertussis was efficiently expressed in Escherichia coli K-12 and Salmonella typhimurium aroA at levels higher than those found in wild-type B. pertussis when the upstream signals of the gene were replaced and the translation initiation region was engineered to optimize translational efficiency. Inclusion of part of the C-terminal FHA open reading frame, whose translation product does not appear to be part of the major secreted species of FHA, was shown to be important in achieving protein expression in both E. coli and S. typhimurium aroA; removal of the downstream gene sequence abolished recombinant FHA production. The levels of expression observed varied widely according to the construct and host bacterium used.

Adhesins, Bacterial↗

PubMed1992

Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase. Investigation of the role of active-site His181.

A system has been developed to allow the convenient production, expression and purification of site-directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised; both the amino acid sequence and crystal structure have been determined and a reaction mechanism has been proposed. However, the molecular basis for catalysis remains poorly understood, with only circumstantial evidence for the roles of most of the active site residues other than His8, which is phosphorylated during the reaction cycle. A vector/host expression system has been designed which allows recombinant forms of phosphoglycerate mutase to be efficiently expressed in yeast with no background wild-type activity. A simple one-column purification protocol typically yields 30 mg pure enzyme/1 l of culture. The active-site residue, His181, which is thought to be involved in proton transfer during the catalytic cycle, has been mutated to an alanine. The resultant mutant has been purified and characterised. Kinetic analysis shows a large decrease (1.6 x 10(4)) in the catalytic efficiency, and an 11-fold increase in the Km for the cofactor 2,3-bisphosphoglycerate. These observations are consistent with an integral role for His181 in the reaction mechanism of phosphoglycerate mutase, probably as a general acid or base.

Binding Sites↗

PubMed2003

Microdystrophin gene therapy of cardiomyopathy restores dystrophin-glycoprotein complex and improves sarcolemma integrity in the mdx mouse heart.

BACKGROUND: More than 90% of Duchenne muscular dystrophy (DMD) patients develop cardiomyopathy, and many die of cardiac failure. Despite tremendous progress in skeletal muscle gene therapy, few attempts have been made to treat cardiomyopathy. Microdystrophin genes are shown to correct skeletal muscle pathological lesions in the mdx mouse model for DMD. Here, we tested the therapeutic potential of adeno-associated virus (AAV)-mediated microdystrophin gene therapy in the mdx mouse heart. METHODS AND RESULTS: AAV was delivered to the newborn mdx mouse cardiac cavity. The procedure was rapid and well tolerated. Efficient expression was achieved in the inner and the outer layers of the myocardium. The ubiquitous cytomegalovirus promoter resulted in substantially higher expression than the muscle-specific CK6 promoter. The therapeutic effects of microdystrophin were evaluated at 10 months after infection. Immunostaining demonstrated extensive microdystrophin expression and successful restoration of the dystrophin-glycoprotein complex. Importantly, AAV-mediated microdystrophin expression improved the sarcolemma integrity in the mdx heart. CONCLUSIONS: We established a simple gene transfer method for efficient and persistent transduction of the mdx mouse heart. AAV-mediated microdystrophin expression restored the critical dystrophin-glycoprotein complex and improved sarcolemma integrity of the mdx heart. Our results revealed the promise of AAV-microdystrophin gene therapy for cardiomyopathy in DMD.

Animals↗

PubMed1998

The Himar1 mariner transposase cloned in a recombinant adenovirus vector is functional in mammalian cells.

Mariner transposons belong to the mariner /Tc1 superfamily of class II, DNA-mediated elements. One of these transposons, Himar1 , isolated from the horn fly, is independent of host-specific factors that would limit transfer between different species, making it an ideal candidate for gene transfer technology development. To determine the activity of Himar1 transposase in mammalian cells, we introduced the Himar1 transposase gene into an adenovirus (Ad) vector under control of the phage T7 RNA polymerase promoter. Mammalian cells infected with the Ad vector carrying the Himar1 gene efficiently expressed the Himar1 transposase in the presence of T7 polymerase. In in vitro inter-plasmid transposition reactions, Himar1 transposase expressed by the Ad vector mediated precise cut-and-paste transposition and resulted in a characteristic duplication of TA at the integration site of the target plasmid. Further studies showed that this transposase was capable of catalyzing transposition between twoplasmids co-transfected into 293T7pol cells, which express T7 RNA polymerase. Combining the integration capability of mariner transposons with the transduction efficiency of Ad vectors is expected to provide a powerful tool for introducing transgenes into the host chromosome.

Adenoviridae↗

PubMed1995

Biochemical and calmodulin binding properties of estrogen receptor binding cyclophilin expressed in Escherichia coli.

Bovine estrogen receptor binding cyclophilin (ERBC), a cyclophilin component of the unactivated estrogen receptor, has been efficiently expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) and purified by single-step chromatography on glutathione-agarose. Thrombin cleavage from GST allowed the isolation of purified, recombinant ERBC. The fusion protein, GST-ERBC, and recombinant ERBC were both characterised for peptidyl prolyl cis-trans isomerase activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate, GST-ERBC demonstrated a kcat/KM value of 5.1 x 10(5) M-1s-1 at 5 degrees C. The isomerase activity was inhibited by cyclosporin A with an IC50 value of 1030 nM. These values indicate that ERBC has a decreased catalytic efficiency and sensitivity to cyclosporin A relative to human cyclophilin. Retention of the GST-ERBC fusion protein on calmodulin-agarose in the presence of Ca2+ and subsequent elution with EGTA has provided evidence that ERBC is a calmodulin-binding protein.

Amino Acid Isomerases↗

PubMed1997

Mutational analysis of the rolA gene of Agrobacterium rhizogenes in tobacco: function of the rolA pre-mRNA intron and rolA proteins defective in their biological activity.

The rolA gene of Agrobacterium rhizogenes contains in its untranslated leader region a spliceosomal intron, which is spliced in Arabidopsis and in Nicotiana tabacum. Expression under the control of the 35S promoter from cauliflower mosaic virus of a rolA gene derivative defective in splicing still causes alterations of growth in transgenic tobacco plants. Splicing of rolA mRNA is required for efficient expression of the rolA phenotype in vivo. Moreover, splicing is required for efficient in vitro translation of the rolA mRNA. In contrast, expression of a 35S-rolA gene derivative with the ATG initiation codon replaced by ATA does not cause any phenotypical alteration. Mutations leading to amino acid substitutions at positions 37 and 40 of the rolA coding region were isolated as null mutants in Arabidopsis plants transgenic for the rolA gene. However, when expressed in tobacco under the control of the 35S promoter, they cause a rolA phenotype reduced in the expressivity of its traits. The molecular characterization of rolA mutants might be useful for understanding the biochemical function of the rolA protein.

Bacterial Proteins↗

PubMed1994

Production of an atrial natriuretic peptide variant that is specific for type A receptor.

Receptor-specific variants of atrial natriuretic peptide (ANP) were selected from libraries of filamentous phage particles that displayed single copies of random ANP mutants fused to gene III protein. These ANP variants were differentially selected by binding to immobilized natriuretic peptide receptor A (NPR-A) over competing receptor C (NPR-C) in solution. This method also selected ANP variants with improved secretion expression in Escherichia coli. Several of the identified mutations were combined to produce an efficiently expressed ANP analog that was as potent as wild-type ANP in stimulating NPR-A guanylyl cyclase activity but resistant to inactivation mediated by NPR-C. Such NPR-A-selective analogs should be useful for correlating the various activities of ANP to the relevant receptor and may also be more potent therapeutics in the targeting of NPR-A.

Amino Acid Sequence↗

PubMed1998

Secretory expression of human albumin domains in Saccharomyces cerevisiae and their binding of myristic acid and an acylated insulin analogue.

Albumin is organized in three homologous domains formed by double loops stabilized by disulfide bonds. Utilizing a secretory expression system based on a synthetic secretory prepro-leader, the three human serum albumin domains were expressed in the yeast Saccharomyces cerevisiae. Human serum albumin domains I and III were efficiently expressed and secreted, indicating that these domains can form independent structural units capable of folding into stable tertiary structures. In contrast, albumin domain II was not secreted and disappeared early in the secretory pathway. Human serum albumin has the ability to bind a large number of small molecule ligands, including fatty acids, presumably due to its structure and structural flexibility. Purified albumin domain III bound myristic acid, whereas purified albumin domain I did not bind myristic acid. A new soluble long-acting insulin an alogue acylated with myristic acid (Markussen J., et al., Diabetologia 39, 281-288, 1996) bound to domain III and bound markedly more weakly to domain I.

Carrier Proteins↗

PubMed1999

Soluble expression of cloned phage K11 RNA polymerase gene in Escherichia coli at a low temperature.

The gene 1 of the Klebsiella phage K11 encoding the phage RNA polymerase was amplified using the polymerase chain reaction of the Pfu DNA polymerase, cloned and expressed under the control of tac promoter in Escherichia coli. Although the gene was efficiently expressed in E. coli BL21 cells at 37 degrees C, most of the K11 RNA polymerase produced was insoluble, in contrast to soluble expression of the cloned T7 RNA polymerase gene. Coexpression of the bacterial chaperone GroES and GroEL genes together did not help solubilize the K11 RNA polymerase. When the temperature of cell growth was lowered, however, solubility of the K11 RNA polymerase was increased substantially. It was found much more soluble when expressed at 25 degrees C than at 30 and 37 degrees C. Thus, the cloned K11 RNA polymerase gene was expressed in E. coli mostly to the soluble form at 25 degrees C. The protein was purified to homogeneity by chromatography using DEAE-Sephacel and Affigel-blue columns and was found to be active in vitro with the K11 genome or a K11 promoter. The purified K11 RNA polymerase showed highly stringent specificity for the K11 promoter. Low-level cross-reactivity was shown with the SP6 and T7 consensus promoters, while no activity shown with the T3 consensus promoter at all.

Base Sequence↗

PubMed1993

Aberrant tissue specific expression of the transgene in transgenic mice that carry the hepatitis B virus genome defective in the X gene.

The control mechanisms for the transgene expression in mice that carry the hepatitis B virus genome defective in the polymerase and X genes were analyzed. Ten lines of transgenic mouse were established, and in seven lines the surface and e antigens were detected in the serum. In transgenic mice from five lines examined, the transgene was markedly expressed in a broad spectrum of tissues including the kidney, heart, brain, muscle and intestine, but only poorly in the liver. In the kidney and heart the 3.5 kb and 2.1 kb mRNAs were expressed, whereas only the 0.8 kb and 4.0 kb mRNAs were detected in the testis and brain, respectively, suggesting that each of the mRNAs was transcribed through a different control mechanism. The surface, e and core antigens accumulated in the kidney and heart. DNA was hypomethylated at a region closely downstream of the enhancer in the liver, kidney and heart, and a DNase I hypersensitive site was detected upstream of the enhancer in these tissues. In the testis, however, the whole transgene was hypomethylated and the DNase I hypersensitive site was closer to the enhancer. These differences may be relevant to the preferential expression of the 0.8 kb mRNA in the testis, but cannot explain the inefficiency of transgene expression in the liver. Our observations suggest that the X protein is required for efficient expression of the viral gene in the liver but not in other tissues.

Animals↗

PubMed2005

Expression and characterization of a thermostable serine protease (TfpA) from Thermomonospora fusca YX in Pichia pastoris.

A serine protease produced by Thermomonospora fusca YX (TfpA) is heat-stable (up to 85 degrees C) and has a broad pH activity range and strong resistance to detergents. The objective of this study was to determine if the methylotropic yeast Pichia pastoris could express TfpA extracellularly. A 1.0-kb DNA fragment (tfpA) encoding the pro-peptide and mature protein of TfpA was cloned into expression vectors pPICZalphaA (inducible) and pGAPZalphaA (constitutive) and introduced into P. pastoris by electroporation. Expression of r-TfpA was greater in the inducible system than in the constitutive one, producing 135 U ml(-1) medium supernatant 6 days after methanol induction. The r-TfpA was not glycosylated (21.7 kDa), and had pH and temperature optima of 8.5 and 80 degrees C, respectively, using azocasein as a substrate. In conclusion, P. pastoris can be used as a host to produce extracellular r-TfpA, and expression efficiency may be improved by optimizing fermentation conditions and modifying factors related to protein expression and stability.

Actinomyces↗