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PubMed1990

27 amino acid residues can be deleted from the N-terminus of human lymphotoxin without impairment of its cytotoxic activity.

In order to study the relationship between activity and structure of human lymphotoxin (hLT, 171 aa), we synthesized the gene (519 bp) for hLT and expressed it in Escherichia coli. Purification of the recombinant hLT from crude extracts was difficult because of the low level of expression of the gene. To improve the yield of the recombinant protein, we prepared five truncated genes for mutant proteins in which 25, 26, 27, 28 and 37 amino acid residues, respectively, were missing from the N-terminus. All of the genes were efficiently expressed and adequate amounts of mutant proteins were synthesized. The proteins were recovered mainly in the supernatant fractions after disruption of cells, with the exception of LT delta 37N, in which 37 residues were absent from the N-terminal region. Cytotoxic activities against mouse fibroblast L929 cells were detected in supernatant fractions that contained these mutant proteins, except in the case of LT delta 28N, which lacks the first amino acid residue conserved in both hLT and human tumour necrosis factor (hTNF). LT delta 27N, which is the smallest of the active proteins, was purified to homogeneity, and its cytotoxic activity was found to be similar to that of recombinant hTNF.

Amino Acid Sequence↗
PubMed1997

Adenoviral vector-mediated interleukin-10 expression in vivo: intramuscular gene transfer inhibits cytokine responses in endotoxemia.

Interleukin-10 (IL-10) is a potent anti-inflammatory/immune cytokine and has received growing attention for its therapeutic potential. To aid therapeutic studies of IL-10 in vivo, a replication-deficient adenoviral vector expressing mouse IL-10 was constructed and characterized. The transgene protein IL-10 was shown to markedly inhibit endotoxin-induced tumor necrosis factor alpha (TNF alpha) production by mouse and rat macrophages in vitro. Intramuscular injection of this vector in mice resulted in efficient expression of transgene mRNA in the muscle and active release of IL-10 protein into the bloodstream. To investigate the therapeutic potential of IL-10 using this vector, endotoxemia was induced by intraperitoneal injection of a sublethal dose of endotoxin. Expression of TNF alpha and IL-6 mRNA in the lung, spleen and heart and the circulating levels of these cytokines markedly increased in endotoxemia. This endotoxin-induced TNF alpha and IL-6 up-regulation was however suppressed in mice expressing IL-10 after intramuscular gene transfer. While cytokine gene expression was inhibited to varying degrees in different organs, a maximal reduction was seen in the lung, thus also indicating the efficacy of systemic IL-10 gene product at multiple tissue sites. Finally, we provided evidence that only when present in abnormally high concentrations in the circulation following intraperitoneal gene delivery, IL-10 by itself had some toxic effects of transient nature, primarily manifested by acute phase reaction and hemostatic disturbance. Thus, our studies demonstrate the usefulness of adenoviral vectors for therapeutic applications of IL-10 in vivo.

Acute-Phase Reaction↗
PubMed1998

Enhanced expression of an antimicrobial peptide sarcotoxin IA by GUS fusion in transgenic tobacco plants.

To enhance the disease resistance of plants expressing a foreign peptide, the gene for sarcotoxin IA, which is an antimicrobial peptide from an insect consisting of 39 amino acid residues, was introduced into tobacco (Nicotiana tabacum) under the control of a high expression promotor via Agrobacterium-mediated transformation. In transgenic plants, sarcotoxin IA mRNA accumulated to detectable levels, however, the amount of the peptide produced was so small that we could scarcely detect it by protein gel blot analysis, probably because of the instability of short peptides in plant cells. To improve the expression efficiency, genes for four types of amino-terminal and carboxyl-terminal translational fusions of sarcotoxin IA together with the GUS gene were introduced into tobacco. In all four types of transgenic tobacco plants, high level transcripts similar to that in the direct expression sarcotoxin IA construct were found. Protein gel blot analysis with both anti-sarcotoxin IA and GUS antibodies showed production of high levels of fusion protein in all transgenic plants. Among them, three types had abnormal membranes and phenotypes, although no such abnormalities were found in transgenic plants in which only sarcotoxin IA was expressed in a secretable form. All together, these results indicated that, for stable and effective expression of a foreign short peptide in transgenic plants, expression as a fusion protein is useful and that secretion of sarcotoxin IA outside of cells is necessary for generation of useful antimicrobial transgenic plants.

Anti-Bacterial Agents↗
PubMed1973

Role of cyclic adenosine 3',5'-monophosphate in the in vivo expression of the galactose operon of Escherichia coli.

Studies of levels of galactokinase in Escherichia coli with mutations affecting synthesis of, or response to, cyclic adenosine 3',5'-monophosphate show that this nucleotide does not play a major role in expression of the galactose operon, causing at most a twofold stimulation. The discrepancy between our in vivo results and the marked stimulation by cyclic adenosine 3',5'-monophosphate in in vitro systems indicates that current cell-free systems lack a factor which allows efficient expression of the galactose operon even in the absence of cyclic adenosine 3',5'-monophosphate or of the binding protein for this nucleotide.

Carbon Isotopes↗
PubMed1989

Expression and purification of biologically active v-sis/platelet-derived growth factor B protein by using a baculovirus vector system.

Malignant transformation induced by simian sarcoma virus is mediated by its v-sis protein, the monkey homolog of the platelet-derived growth factor (PDGF) B chain. By use of an appropriately engineered baculovirus expression vector, the v-sis protein was expressed in the insect cell line Spodoptera frugiperda (Sf9) at a level 50- to 100-fold higher than that observed with overexpression in mammalian-cell transfectants. The sis protein produced by Sf9 cells underwent processing similar to that observed in mammalian cells, including efficient disulfide-linked dimer formation. Moreover, the recombinant sis protein was capable of binding PDGF receptors and inducing DNA synthesis as efficiently as PDGF-B synthesized by mammalian cells. A significant fraction of sis protein was released from Sf9 cells, which made possible a one-step immunoaffinity purification to near homogeneity with a 40% recovery of biological activity. These results demonstrate that a protein whose normal processing requires both intrachain and interchain disulfide-bridge formation can be efficiently expressed in a biologically active form in insect cells by using a baculovirus vector system.

Amino Acid Sequence↗
PubMed2026

Preclinical evaluation of AL-001, a gene therapy for wet age-related macular degeneration.

BACKGROUND: Frequent intravitreal administration of antivascular endothelial growth factor Vascular endothelial growth factor agents remains a major limitation in the management of wet age-related macular degeneration (wAMD). This study evaluated whether suprachoroidal delivery of an engineered recombinant adeno-associated viral (rAAV)-aflibercept vector could achieve sustained, targeted expression with improved efficacy and safety compared with intravitreal administration. METHODS: AL-001, an engineered rAAV vector expressing aflibercept, was developed and characterized. Its expression profile was first assessed in New Zealand white rabbits following suprachoroidal space (SCS) injection. Efficacy, pharmacokinetics, and safety were then evaluated in a nonhuman primate model of laser-induced choroidal neovascularization (CNV), comparing SCS and intravitreal (IVT) administration routes. RESULTS: AL-001 efficiently expressed aflibercept in relevant ocular cells in vitro. In rabbits, SCS administration produced sustained aflibercept levels in ocular tissues. In the nonhuman primate CNV model, a single SCS injection of AL-001 showed favorable efficacy to IVT injection and a notable mild inflammatory response. At week 4, grade IV lesion incidence was 0% (0/48) after SCS administration versus 14.3% (6/42) after IVT administration (absolute difference, -14.3 percentage points; 95% CI, 3.7%-27.8%; P = 0.0258). Throughout follow-up, mean leakage area and grade IV lesion incidence remained 0 with SCS, versus IVT peaks of approximately 0.3 mm2 and 33.0%, respectively, declining to 0.03 mm2 and 2.0% by day 100. Both the medium and high doses decreased pathological vascular leakage and subretinal hyperreflective material. Vector administration preceded laser-induced CNV modeling, demonstrating that sustained intraocular aflibercept expression in the retina and choroid provided durable antiangiogenic protection. Pharmacokinetic analysis confirmed distinct ocular exposure profiles between routes, with viral genomes confined predominantly to the injected eye and no significant systemic accumulation. AL-001 was well tolerated, without sustained intraocular pressure elevation or severe ocular inflammation, and only mild-to-moderate treatment-emergent adverse events. Low pre-existing anti-AAV2 immunity and time-dependent neutralizing antibody responses postdosing, informing a translational model for patient stratification and redosing feasibility. CONCLUSION: Suprachoroidal administration of AL-001 is well tolerated and provides durable, targeted aflibercept expression with pronounced antiangiogenic efficacy. These results support AL-001 as a promising, long-acting therapeutic candidate for wAMD.

AAV↗
PubMed1999

Transcription of the beta-galactoside alpha2,6-sialyltransferase gene (SIAT1) in B-lymphocytes: cell type-specific expression correlates with presence of the divergent 5'-untranslated sequence.

A single gene, SIAT1, encodes ST6Gal I, the sialyltransferase that mediates transfer of alpha2,6-linked sialic acids to Galbeta1, 4GlcNAc termini of N-linked glycoproteins. In vivo, multiple SIAT1 mRNA forms, differing only in the 5'-untranslated region, are expressed in a tissue-specific manner. This mRNA heterogeneity has been attributed, at least in part, to transcription from a number of physically distinct promoter regions. In mature B-lymphocytes, SIAT1 transcription initiates at P2, a regulatory region known to function only in B-lineage cells. Bacterial chloramphenicol acetyltransferase (CAT) under the control of the P2 region encompassing 415 bp 5'- and 125 bp 3' of the transcriptional initiation site is efficiently expressed in Louckes, a mature B-lymphoblastoid cell line. In contrast, CAT expression in Reh, a T-null/B-null precursor line, and in HepG2, a hepatoma line, are 14-fold and >25-fold less than in Louckes, respectively. The data is consistent with the presence of cis -acting regulatory elements residing both 5' and 3' of the P2 transcriptional initiation site. At least 370 bp of 5'-flanking sequence, coinciding with the inclusion of AP2 and NF-kappaB sites, is necessary for high level expression in Louckes. Exon sequences 3' of the transcription start site are also important for expression. A segment from(+)32 to(+)125 (position(+)1 is transcription start site) is capable of exerting promoter-like activity in Louckes, but not in Reh or HepG2. CAT expression by P2 is negligible in Reh cells. However, enhanced CAT activity is not accompanied by elevated mRNA levels. This observation is consistent with the relief of translational restraints imposed by the(+)32 to(+)125 region. Together, the data demonstrate that efficient and cell-specific transcription regulation in mature B lymphocytes is contained in a 495 bp P2 segment that is comprised of 370 bp of 5'-flanking region and 125 bp of transcribed region of Exon X.

5' Untranslated Regions↗
PubMed1999

Comparison of the effectiveness of adenovirus vectors expressing cyclin kinase inhibitors p16INK4A, p18INK4C, p19INK4D, p21(WAF1/CIP1) and p27KIP1 in inducing cell cycle arrest, apoptosis and inhibition of tumorigenicity.

Cell cycle regulatory proteins are important candidates for therapeutic tumour suppressors. Adenovirus vectors were constructed to overexpress cyclin kinase inhibitors p16INK4A, p18INK4C, p19INK4D, p21(WAF1/CIP1) and p27KIP1 under the control of the murine cytomegalovirus immediate early gene promoter. These vectors directed the efficient expression of each of the cyclin kinase inhibitors and induced growth arrest, inhibited DNA synthesis, and prevented phosphorylation of the retinoblastoma protein (pRb) in cell lines expressing functional pRb. In pRb-deficient cells, expression of the cyclin kinase inhibitors was not effective in inhibiting DNA replication or growth arrest. Interestingly, three of the cyclin kinase inhibitors, p16, p18 and p27 were found to induce apoptotic death in transduced HeLa and A549 cells. When the vectors were tested for their ability to inhibit tumorigenicity in a polyomavirus middle T antigen model of murine breast carcinoma, expression of the cyclin kinase inhibitors resulted in a delay in tumour formation that varied from several weeks for the p19 expressing vector to greater than 25 weeks for the p27 expressing vector. When tumours were injected directly with the adenovirus vectors expressing the cyclin kinase inhibitors, only treatment with the vector expressing p16 resulted in a delay in tumour growth.

Adenocarcinoma↗
PubMed1999

Adenovirus-mediated expression of mutant DRPLA proteins with expanded polyglutamine stretches in neuronally differentiated PC12 cells. Preferential intranuclear aggregate formation and apoptosis.

To investigate the molecular mechanisms of neurodegeneration caused by expanded CAG repeats in dentatorubral-pallidoluysian atrophy (DRPLA), an autosomal dominant neuro degrees enerative disorder caused by unstable expansion of a CAG trinucleotide repeat in the DRPLA gene on 12p13.31, we established an efficient expression system for truncated and full-length DRPLA proteins with normal or expanded polyglutamine stretches in neuronally differentiated PC12 cells and fibroblasts using an adenovirus expression system. Although aggregate body formation was observed both in neuronally differentiated PC12 cells and in fibroblasts expressing truncated DRPLA proteins with Q82, >97% ( n = 3) of neuronally differentiated PC12 cells showed intra-nuclear inclusions, while only 31 21% ( n = 3) of fibro-blasts had intranuclear inclusions at 3 days after infection. The percentage of apoptotic cells was significantly higher in neuronally differentiated PC12 cells expressing the truncated DRPLA protein with Q82 than in fibroblasts, suggesting the possibility that intranuclear aggregate bodies are formed preferentially in neuronally differentiated PC12 cells and that these cells are more vulnerable than fibroblasts to the toxic effects of expanded polyglutamine stretches in the DRPLA protein. When the full-length DRPLA protein with Q82 was expressed, aggregate bodies were found exclusively in the nuclei of the neuronally differentiated PC12 cells, while they were found in the cytoplasm of fibroblasts. Despite the presence of aggregate bodies, apoptosis was not induced by expression of the full-length DRPLA protein with Q82 in either neuronally differentiated PC12 cells or fibroblasts, suggesting that the presence of intranuclear aggregate bodies is in itself not necessarily toxic to cells.

Adenoviridae↗
PubMed1989

Cloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene.

A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in 1,2-dichloroethane metabolism were isolated. The haloalkane dehalogenase gene dhlA was subcloned, and it was efficiently expressed from its own constitutive promoter in strains of a Pseudomonas sp., Escherichia coli, and a Xanthobacter sp. at levels up to 30% of the total soluble cellular protein. A 3-kilobase-pair BamHI DNA fragment on which the dhlA gene is localized was sequenced. The haloalkane dehalogenase gene was identified by the known N-terminal amino acid sequence of its product and found to encode a 310-amino-acid protein of molecular weight 35,143. Upstream of the dehalogenase gene, a good ribosome-binding site and two consensus E. coli promoter sequences were present.

Amino Acid Sequence↗
PubMed2003

Molecular determinants responsible for differential cellular distribution of G protein-gated inwardly rectifying K+ channels.

Activation of the heteromeric G protein-gated inwardly rectifying K(+) channel (GIRK) GIRK1 and GIRK4 subunits gives rise to I(KACh), which controls excitability in atrial tissue. Although homomeric GIRK4 channels localize to the plasma membrane and display moderate function, GIRK1 channels fail to localize to the cell surface and do not exhibit significant function as homomers. Using oocytes to express GFP-tagged GIRK1 and GIRK4 and chimeras between these two proteins, we have identified two regions, one in the proximal C terminus and another in the distal N terminus that are critical for their subcellular localization. Replacement of both of these regions in GIRK1 with corresponding regions from GIRK4 was required for efficient expression of GIRK1 on the plasma membrane. Replacement of either region by itself was ineffective. The distal N terminus and proximal C terminus have been previously suggested to play important roles in ER-export and subunit co-assembly respectively in this family of channels. Our data indicate for the first time that both of these regions need to work in concert to mediate efficient targeting of these channels to the plasma membrane.

Animals↗
PubMed1987

A transcriptional activator is located in the coding region of the yeast PGK gene.

Expression of heterologous genes from the PGK promoter on high copy number plasmids in yeast is relatively poor compared to the intact PGK gene because of low steady-state RNA levels. In this paper we show that low levels of heterologous RNA are not due to instability of mRNA but result from inefficient transcription due to a defect in RNA synthesis. A comparison of RNA levels from homologous and heterologous transcription units allowed the identification of a positive activator for transcription within the PGK coding region which is required for efficient expression of the PGK gene. Deletion of this region, the "downstream activator sequence", causes a six to ten fold reduction in transcriptional efficiency from the PGK 5' noncoding region.

Base Sequence↗
PubMed1991

Gene synthesis, expression in Escherichia coli, purification and characterization of the recombinant bovine acyl-CoA-binding protein.

A synthetic gene encoding the 86 amino acid residues of mature acyl-CoA-binding protein (ACBP), and the initiating methionine was constructed. The synthetic gene was assembled from eight partially overlapping oligonucleotides. Codon usage and nucleotides surrounding the ATG translation-initiation codon were chosen to allow efficient expression in Escherichia coli as well as in yeast. The synthetic gene was inserted into the expression vector pKK223-3 and expressed in E. coli. In maximally induced cultures, recombinant ACBP constitutes 12-15% of total cellular protein. A fraction highly enriched for recombinant ACBP was obtained by extracting induced E. coli cells with 1 M-acetic acid. Recombinant ACBP was purified to homogeneity by successive use of gel-filtration chromatography, ion-exchange chromatography and reverse-phase h.p.l.c. Recombinant ACBP differed from native ACBP by lacking the N-terminal acetyl group. The acyl-CoA-binding characteristics of recombinant ACBP did not differ from those of native ACBP, and the two proteins showed the same ability to induce medium-chain acyl-CoA synthesis by goat mammary-gland fatty acid synthetase. It was concluded that the N-terminal acetyl group is not important for acyl-CoA binding.

Acyl Coenzyme A↗
PubMed1985

Expression of Plasmodium falciparum circumsporozoite proteins in Escherichia coli for potential use in a human malaria vaccine.

The circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum may be the most promising target for the development of a malaria vaccine. In this study, proteins composed of 16, 32, or 48 tandem copies of a tetrapeptide repeating sequence found in the CS protein were efficiently expressed in the bacterium Escherichia coli. When injected into mice, these recombinant products resulted in the production of high titers of antibodies that reacted with the authentic CS protein on live sporozoites and blocked sporozoite invasion of human hepatoma cells in vitro. These CS protein derivatives are therefore candidates for a human malaria vaccine.

Animals↗
PubMed1992

Primary structure and in vitro expression of the N. crassa phosphoglycerate kinase.

The primary structure of 3-phosphoglycerate kinase (PGK) from Neurospora crassa was determined by sequencing a full-length cDNA. The deduced 418 amino acids protein shows a considerable identity to PGKs of other organisms with all the residues thought to be important for the function of the yeast enzyme conserved. The cloned PGK cDNA could be efficiently expressed in vitro resulting in a product with the expected molecular weight.

Amino Acid Sequence↗
PubMed2004

Methanol induction optimization for scFv antibody fragment production in Pichia pastoris.

Fibronectin splice variant ED B (extracellular domain B) is a promising marker for angiogenesis in growing solid tumors. Currently, recombinant antibodies against ED B are being investigated concerning their potential use, for either therapeutic or diagnostic purposes. Single-chain antibody fragments directed against the ED B can be efficiently expressed in Pichia pastoris; thus, a recombinant strain of the methylotropic yeast P. pastoris was used for this work. Three different forms of scFv antibody fragment are found in the supernatant from this fermentation: covalent homodimer, associative homodimer, and monomer. Both homodimeric forms can be converted to the monomeric form (under reducing conditions) and be efficiently radiolabeled, whereas the monomeric form of scFv already present in the supernatant cannot. It was also found that the fraction of protein in the monomeric form is highly dependent on the mode of induction rather than scFv concentration. This suggests that the monomeric form of the scFv present in the supernatant might be a result of events occurring at the expression, secretion, or folding level. A high cell density fermentation protocol was developed by optimizing methanol induction, yielding the highest scFv antibody fragment production rate and product quality; cell concentration at the induction point and specific methanol uptake rate were found to be the most important control variables. A decrease in specific methanol uptake rate led to a higher specific production rate for the scFv antibody fragment (5.4 microg g(cell) h(-1)). Product quality, i.e., percentage of product in a homodimeric form, also increased with the decrease in methanol uptake rate. Furthermore, the volumetric productivity depended on cell concentration at the induction point, increasing with the increase of cell concentration up to 320 g L(-1) wet cell weight (WCW). The reduction of the methanol feeding rate for induction, and consequently of the oxygen uptake rate, have important consequences for optimizing product titers and quality and thus on the scale-up of this production process; hence one of the major limitations upon high cell density cultivation in bioreactors is keeping the high oxygen transfer rate required. From the results obtained, a scale-up strategy was developed based on the available oxygen transfer rates at larger scales, allowing the definition of the optimum biomass concentration for induction and methanol feeding strategy for maximization of product titer and quality.

Cell Division↗
PubMed1987

Functional characterization of X. laevis U5 snRNA genes.

Xenopus laevis U5 snRNA genes are found in several genomic arrangements, represented by a predominant tandem repeat of 583 bp and other minor repeats. Several copies of the major tandem repeat have been cloned and expressed in Xenopus oocytes. The transcripts assemble into U5 snRNPs which are recognized by anti-Sm antibodies. We have identified functional elements in the U5 gene promoter. Although similar in organization to other U snRNA gene promoters, U5 contains significant differences and is more efficiently expressed than the Xenopus U2 gene in oocytes. The proximal sequence element (PSE), although homologous to a mammalian consensus for this region (Skuzeski et al., 1984), does not resemble the previously characterized Xenopus U1 and U2 PSEs closely in sequence. The ATGCAAAT (octamer) part of the distal sequence element (DSE 1) is found in U5 in the orientation opposite to that in U1 and U2 gene promoters. DNase I protection experiments led to the identification of a third element (DSE 2), situated close to the octamer motif. Analysis of deletion mutants showed that both DSE 1 and 2 are essential parts of the U5 gene enhancer, and provides evidence that U snRNA enhancers are complex structures consisting of more than one site of DNA-factor interaction.

Animals↗
PubMed1996

Deletion analysis of promoter elements of the Aspergillus oryzae agdA gene encoding alpha-glucosidase.

The nucleotide sequence of a 1.5-kb fragment of the promoter region of the Aspergillus oryzae agdA gene encoding alpha-glucosidase was determined. A comparison with the promoter regions of other Aspergillus amylase genes indicated that there are three highly conserved sequences, designated Regions I, II and III, located at -670 nt, -596 nt and -544 nt relative to the start codon, respectively. The function of these consensus sequences in the agdA promoter was investigated by deletion analysis of a promoter fusion with the Escherichia coli uidA gene, using the niaD homologous-transformation system. Deletion of the upstream half of Region III (IIIa; -544 to -529) resulted in a more than 90% reduction in GUS activity and abolished maltose induction, suggesting that Region IIIa is a functionally essential element for high-level expression and maltose induction. Deletion of Region I and the downstream half of Region III (IIIb; -521 to -511) resulted in a significant reduction in GUS activity, but did not affect maltose induction. This suggested that these two elements most likely contain sequences involved in efficient expression in cooperation with Region IIIa. In addition, deletion of a 340-bp region between Region IIIb and the putative TATA box resulted in a 2-fold increase in activity.

Aspergillus oryzae↗