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At least 37 records · Page 2Linked to original sources
PubMed1990

Efficient expression of human beta-interferon in Namalwa KJM-1 cells adapted to serum-free medium by a dhfr gene coamplification method.

We previously reported the expression of human beta-interferon (beta-IFN) (Miyaji et al., 1989) and human lymphotoxin (Miyaji et al., 1990) in Namalwa KJM-1 cells adapted to serum-free medium. To establish an efficient gene expression system, a dihydrofolate reductase (dhfr) gene coamplification method was applied to this cell line. A beta-IFN expression plasmid was introduced with a dhfr expression plasmid into KJM-1 and methotrexate (MTX)-resistant derivatives were selected by a stepwise increase of MTX concentration. Among them, derivatives which showed higher expression levels of beta-IFN than that achieved by the parental transformants were obtained, suggesting that a dhfr gene coamplification method can be used for efficient expression of foreign genes in KJM-1 which contains endogenous dhfr genes. Then, an improved beta-IFN expression vector was constructed, which contains a dhfr transcription unit. This plasmid was introduced into KJM-1 and then, MTX-resistant derivatives were selected. Among them, the highest producer, clone 40-10-24, secreted beta-IFN at a level as high as 5 micrograms/ml, which is about 100-fold higher than that obtained by the G418-resistant parental transformants. In addition, beta-IFN produced by recombinant KJM-1 cells had the same molecular weight of that produced by fibroblasts.

Blotting, Western↗
PubMed1992

Involvement of a possible chaperonin in the efficient expression of a cloned CryIIA delta-endotoxin gene in Bacillus thuringiensis.

The Bacillus thuringiensis cryIIA delta-endotoxin gene is found as the third-gene in a three-gene operon, with a sporulation-dependent promoter lying upstream of the first gene, orf1. We show here that the polypeptide product of the middle gene (orf2) is required for efficient expression of the toxin gene. In the absence of a functional ORF2 polypeptide the toxin does not form the crystalline inclusions characteristic of other known Bacillus thuringiensis toxins. We discuss the importance of this finding with respect to the possible role of chaperonins in the crystallization of these proteins.

Amino Acid Sequence↗
PubMed1989

Efficient expression and characterization of isolated structural domains of yeast phosphoglycerate kinase generated by site-directed mutagenesis.

The two domains of yeast phosphoglycerate kinase were produced by recombinant techniques. The N-domain was obtained by the introduction of a termination codon at the position coding for Phe185, and the C-domain by a deletion in the gene of the coding sequence between Ser1 and Leu186. Both domains were efficiently expressed in yeast, the level for the C-domain being greater than that for the N-domain. Both domains were found to have a quasi-native structure; the C-domain retained its ability to bind nucleotides. Small local differences were detected in domain structure compared to that in the whole enzyme, probably due to the lack of interdomain stabilizing interactions. Nevertheless, such an approach provides direct evidence for independent folding of domains in a two-domain protein.

Cloning, Molecular↗
PubMed1992

Importance of the carboxyl terminus of human thyroid peroxidase in the efficient expression of the protein in eukaryotic cells.

Carboxyl terminal truncation of membrane-associated human thyroid peroxidase (hTPO), with the elimination of its single membrane-spanning and short intracytoplasmic regions, generates a soluble, secreted, enzymatically active protein (amino acids 1-848). In order to determine the effects of further carboxyl terminal deletions on the expression of hTPO, Chinese hamster ovary cells were stably transfected with plasmids constructed to express amino acids 1-771, 1-636, 1-539 and 1-382 of the 933 amino acid TPO protein, respectively. Unlike hTPO1-848, the more severely truncated TPO mutant proteins could not be detected in conditioned media by polyclonal anti-TPO antibodies. Using detergent-solubilized microsomal proteins from these cells, very low levels of hTPO1-771 (approximately 90 kDa), but not the more extensive deletion mutations, were detected by these anti-TPO antibodies. Confirmation of the loss of efficient expression of more severely truncated hTPO was obtained using a anti-hTPO monoclonal antibody with an epitope near the amino terminus and which recognizes only the denatured protein. The mRNA for all hTPO mutants was detected in the stably-transfected Chinese hamster ovary cells. In summary, the present study indicates that a largely intact extracellular portion of hTPO is required for expression in eukaryotic cells.

Animals↗
PubMed2025

Efficient Expression of Oropouche Virus Nonstructural Proteins NSs and NSm.

Oropouche fever, a mosquito- or midge-borne emerging zoonotic disease endemic to South and Central America, manifests as a dengue-like acute febrile illness with occasional occurrences of meningitis or meningoencephalitis. The causative agent, Oropouche virus (OROV), belongs to the genus Orthobunyavirus within the family Peribunyaviridae. Its tripartite negative-sense RNA genome comprises small (S), medium (M), and large (L) segments, encoding structural N, Gn/Gc, and L proteins, respectively. Additionally, the S- and M-segments encode nonstructural proteins: NSs and NSm, which may act as virulence factors. OROV NSs functions as an interferon antagonist with an unknown mechanism, while the roles of OROV NSm remain elusive. This chapter introduces efficient expression systems for OROV NSm and NSs proteins. Validating the presence of a signal peptide at the N-terminus of NSm protein is essential for its expression. Furthermore, expressing OROV NSs protein independently of an RNA polymerase II promoter is crucial to prevent restricted gene expression, potentially caused by NSs inhibiting cellular RNA polymerase II, as observed in closely related bunyavirus NSs proteins. These protein expression strategies offer insights into the molecular characterization of OROV NSm and NSs proteins, facilitating a deeper understanding of their virulence mechanisms.

Viral Nonstructural Proteins↗
PubMed1987

Molecular analysis of the promoter region of the Escherichia coli K-12 phoE gene. Identification of an element, upstream from the promoter, required for efficient expression of phoE protein.

The phoE gene of Escherichia coli codes for an outer membrane pore protein whose expression is induced under phosphate limitation. The promoter of this gene contains a 17 base-pair fragment, designated a pho box, which is present also in other phosphate-controlled promoters. The mRNA start site was determined and found to be located downstream from the pho box, such that this element is located in the -35 region of the phoE promoter. A set of promoter deletions was generated in vitro and analysis of these deletions revealed that sequences upstream from the pho box are required for the efficient expression of phoE. The required upstream region is located (in part) between positions -106 and -121 relative to the mRNA start site, and contains sequences homologous to a pho box and a correctly spaced Pribnow box, but in the reversed orientation relative to the regular -35 and -10 regions. A proper spacing between this upstream region and the -35 region appears to be important, since an oligonucleotide insertion in the intervening region interferes with phoE expression. By cloning the upstream region in a lacZ operon fusion vector, a weak phosphate limitation-inducible promoter activity could be detected.

Bacterial Outer Membrane Proteins↗
PubMed1991

A cluster of four Sp1 binding sites required for efficient expression of the human insulin receptor gene.

Fragments of 5'-flanking sequences of the human insulin receptor gene were analyzed in transient expression assays after transfection of cell lines with expression assays after transfection of cell lines with an improved low background chloramphenicol acetyl-transferase vector system pSVOOCAT (Araki, E., Shimada, F., Shichiri, M., Mori, M., and Ebina, Y. (1988) Nucleic Acids Res. 16, 1627). Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions and insertions of the promoter of HIR gene into CHO and COS cells indicated that the region between -629 and -1 (initiator ATG is +1) is sufficient for maximal promoter activity. The DNA element of the cluster of four G-C boxes (-593 to -618) enhanced the transcription, examined by the low background pSVOOCAT vector system in vivo. DNase I footprinting and gel retardation experiments using partially purified LacZ-Sp1 hybrid proteins showed that the transcription factor Sp1 can bind to the cluster of the four G-C boxes of the promoter. Thus, the efficient expression of the human insulin receptor gene possibly requires the binding of transcriptional factor Sp1 to four G-C boxes located -593 to -618 base pairs upstream of the ATG translation initiation codon.

Animals↗
PubMed1992

Interferon-alpha but not -beta genes require de novo protein synthesis for efficient expression in human monocytes.

Monocytes produce interferon-alpha (IFN)-alpha) and -beta when human peripheral blood mononuclear cells (PBMCs) are stimulated in vitro by Sendai virus (SV). We found that about 70% of the IFN-producing cells (IPCs) expressed both IFN-alpha and -beta mRNA; the rest expressed only IFN-beta mRNA. In the presence of the protein synthesis inhibitor cycloheximide (CHX), the frequency of IFN-alpha mRNA-containing cells, measured after 6h, was decreased by 85-90%. Results of nuclear run-on transcription assays showed that CHX inhibited IFN-alpha gene expression. The frequency of IFN-beta mRNA-containing cells was not reduced by CHX. Actually, a threefold increase was observed at the lower CHX concentrations. Studies on the kinetics of IFN-alpha/beta mRNA induction showed that CHX accelerated the appearance of IFN-beta mRNA-containing cells, increased IFN-beta mRNA levels, and delayed the normally occurring post-inductional decrease of IFN-beta mRNA. Unexpectedly, an initially normal or even accelerated IFN-alpha mRNA response was seen in the presence of CHX during the first 3-4 h after SV stimulation. This occurred in a small proportion of the potential IPCs. However, CHX prevented the subsequent marked increase of IFN-alpha mRNA levels. Preincubation of PBMCs for 6 h in conditioned medium (CM) containing IFN and other cytokines prevented the CHX-mediated inhibition of IFN-alpha mRNA. Without preincubation this was not seen. The preincubation in CM caused an accelerated appearance of IFN-alpha mRNA, resembling that of IFN-beta mRNA. The results suggest that IFN-alpha and -beta genes are differentially regulated in the same monocytes, the former requiring de novo synthesis of intracellular protein(s) for efficient expression.

Blotting, Northern↗
PubMed1988

Efficient expression of retroviral vector-transduced human low density lipoprotein (LDL) receptor in LDL receptor-deficient rabbit fibroblasts in vitro.

Familial hypercholesterolemia is caused by a genetic deficiency of the low density lipoprotein (LDL) receptor. The Watanabe heritable hyperlipidemic (WHHL) rabbit, which is also defective in LDL receptor activity, provides an excellent animal model of homozygous familial hypercholesterolemia. As a step toward development of effective gene therapy for familial hypercholesterolemia, we have constructed a transmissible retroviral vector containing a full-length human cDNA for the LDL receptor. WHHL fibroblasts infected in vitro expressed the human receptor efficiently, as indicated by RNA and ligand blotting studies. Infected fibroblasts bound and degraded a monoclonal antibody specific for the human LDL receptor (IgGC7) in a manner comparable to that seen with normal human fibroblasts. Human LDL was also degraded by infected WHHL cells and promoted cholesterol esterification to the same degree as seen in normal human fibroblasts. Although technical problems remain to be solved, these studies show that, in principle, gene therapy may be possible for familial hypercholesterolemia patients.

Animals↗
PubMed1986

Use of a high-efficiency expression vector to isolate cDNA clones for factor H and map their positions within the molecule.

A human liver cDNA library, cloned in a novel high-efficiency bacterial expression vector (PEX), was screened with an affinity-purified antibody to human factor H. Four distinct cDNA clones, H-2, H-40, H-46 and H-49, were identified. Of these, H-2 also reacted with two monoclonal antibodies to H, MAH-4 and OX-24, which were previously shown to recognize the 38,000 N-terminal tryptic fragment of H, carrying the binding site for C3b. By using polyclonal antibodies specific for the domains in H coded for by these cDNA-clones, it could be established that H-2 codes only for the 38,000 N-terminal tryptic fragment of H, whereas H-40, H-46 and H-49 are derived from the 142,000 C-terminal fragment of H. By subcloning H-2 the epitope for OX-24 could be localized as being coded near the central Sma-site of H-2.

Antibodies↗
PubMed1985

Comparison of the transient late region expression of SV40 DNA and SV40-based shuttle vectors: development of a new shuttle vector that is efficiently expressed.

Transient expression of an SV40-based shuttle vector similar in design to commonly used vectors is shown to be inefficient when compared to expression of SV40 viral DNA. To eliminate this problem, we have designed and constructed a new vector, pSVPiC, which contains a minimal noninterfering plasmid component, the 885-bp plasmid PiAN7, and two SV40 promoter/origin regions. Transient expression of the SV40 late region from pSVPiC is much more efficient than that from previously used vectors and even more efficient than that from SV40 viral DNA. When the gene for rabbit beta-globin is placed in the late region of pSVPiC, it is also expressed at high levels, indicating that this shuttle vector is generally useful for expressing eukaryotic genes.

DNA Replication↗
PubMed1992

A combination of upstream and proximal elements is required for efficient expression of the mouse renin promoter in cultured cells.

Renin, a key enzyme controlling blood pressure, is produced mainly in the kidney. To identify the transcriptional regulatory elements of the mouse Ren-1c gene, the promoter regions were fused to the CAT reporter gene and transfected into embryonic kidney-derived 293 cells and four extrarenal cell lines, HeLa, HepG2, HT1080 and NIH3T3 cells. Transient transfection assay showed that sequences from -365 to +16 of the renin gene could direct transcription of the CAT hybrid gene only in 293 cells. Deletion analysis identified two transcriptionally active regions; the renin upstream-promoter element (RU-1 element; position -224 to -138) and the renin proximal-promoter element (RP-2 element; position -75 to -47). Although the RU-1 element functioned as an activator, depending on its orientation, it failed to trans-activate the renin promoter when the RP-2 element was deleted. By contrast, the proximal element alone exhibited a weak trans-activator property. Gel shift assay identified RU-1 element-binding factors in both 293 and HeLa cells, whereas 293 cell-dominant factors were shown to bind only to RP-2 element. Therefore, both RU-1 and RP-2 elements were found to be necessary for efficient CAT expression from the renin promoter in 293 cells, suggesting that activation of the Ren-1c promoter requires combined action between cell type-dominant and ubiquitous nuclear factors.

Animals↗
PubMed1992

Functional high efficiency expression of cloned leukocyte chemoattractant receptor cDNAs.

Human kidney 293 TSA cells were transfected by a calcium phosphate method with human formylpeptide and C5a receptor cDNAs with high efficiency. Formylpeptide receptor positive transfectants expressed a total of 968,000 +/- 34,000 receptors per cell with two affinity states (Kds of ca. 0.43 nM and 39 nM), which in the presence of 100 microM GTP gamma S decreased by ca. 4-fold the number of high-affinity sites. The ligand binding pharmacology of cloned and expressed formylpeptide receptors were indistinguishable from endogenous receptors on human neutrophils. Expressed formylpeptide and C5a receptors were functionally active in mobilizing intracellular calcium via a pertussis toxin sensitive mechanism with an ED50 for formylpeptide of ca. 0.5-1.0 nM. This expression system, in which receptor expression can be monitored by flow cytometric methods and in which intracellular calcium responses are measurable, unlike in the more popular COS-7 cell expression system, will provide a useful basis for the analysis of chemoattractant receptor structure-function relationships.

Base Sequence↗
PubMed1992

An efficient expression system for a variant form of the cytotoxic protein alpha-sarcin in Escherichia coli.

An efficient Escherichia coli system for the production of a variant form of the cytotoxic protein alpha-sarcin (delta Ala 1) has been constructed. cDNA encoded alpha-sarcin lacking N-terminal alanine was ligated with the bla signal peptide sequence (the signal sequence of E. coli beta-lactamase localizes the protein in the periplasm) and was inserted into an inducible bacterial expression vector pKTN2-2. When the plasmid introduced into E. coli was expressed in the presence of IPTG, the recombinant product accumulated in the periplasmic space. The product was purified by Affi-Gel Blue followed by Bio-Rex 70 column chromatography. Recombinant alpha-sarcin (delta Ala 1) displayed the properties similar to those of authentic alpha-sarcin isolated from Aspergillus giganteus with respect to its molecular mass and enzymatic activity in ribosomal inactivation. The amount of alpha-sarcin variant produced in the system was estimated to be 1.2 mg/l of culture.

Amino Acid Sequence↗
PubMed1988

Short-term efficient expression of transfected DNA in human hematopoietic cells by electroporation: definition of parameters and use of chemical stimulators.

The efficiency of DNA transfer into human hematopoietic cells by electroporation was investigated and compared to conventional transfection procedures. Important parameters of electroporation were optimized in human erythroleukemia cells using the chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) gene linked to the cytomegaloviral enhancer-promoter. In addition, selected chemicals with different modes of action were studied for their ability to aid DNA entry and gene expression in this system, and several were found to enhance gene transfection by electroporation in a significant manner. Using these chemical stimulators, many but not all human and mouse suspension cultures tested were successfully electroporated by the Baekon 2000 instrument. From these studies, it appears that electroporation can be enhanced by chemical additives. Because of its efficiency, reproductivity, and convenience electroporation is an attractive method of gene transfer in human hematopoietic cells.

Acetyltransferases↗
PubMed1992

Nonreplicating vaccinia vector efficiently expresses recombinant genes.

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

Animals↗
PubMed1987

The human alpha-1-antitrypsin gene is efficiently expressed from two tissue-specific promotors in transgenic mice.

Alpha 1-antitrypsin (alpha 1AT) present in large amounts in human serum and synthesized predominantly in hepatocytes, is the most abundant protease inhibitor and alpha 1AT mutant proteins are associated with hereditary disorders. To investigate the regulation of the normal human alpha 1AT gene, we have microinjected fertilized mouse eggs with a 17.5 kb DNA fragment containing the entire gene with 7 kb 5' and 0.3 kb 3' flanking sequences. We show that this DNA fragment contains all the information for efficient, accurate and tissue-specific expression. High serum concentration of the human protein was found in three independent transgenic mouse lines. The human alpha 1AT RNA is transcribed efficiently in liver, kidney and macrophages and we demonstrate that two different promoters are used for the expression in liver and macrophages of the transgenic mice.

Animals↗
PubMed1988

Construction and characterization of a retroviral vector demonstrating efficient expression of cloned cDNA sequences.

We describe the construction and properties of a retroviral expression vector, designated pMV-7, designed to transfer unselected cDNAs and produce their encoded proteins in recipient cells. The vector is flanked by the long terminal repeats (LTRs) of the Moloney murine sarcoma virus (MSV) and contains the selectable drug resistance gene neo under the regulation of the herpes simplex virus (HSV) thymidine kinase (tk) promoter. Unique Eco RI and Hind III sites facilitate the introduction of sequences whose transcription is regulated by the 5' LTR. We have inserted cDNAs encoding: (i) the human lymphocyte antigen T4, (ii) the human lymphocyte antigen T8, and (iii) the murine hypoxanthine-guanine phosphoribosyl transferase (HPRT), into the pMV-7 vector. These constructions were used to transduce recipient cells to the neo+ phenotype. In each case, functional assays demonstrated that 65-92% of the neo+ clones produced the appropriate protein encoded by its corresponding cDNA. These clones were characterized further by analyzing the expression of vector-regulated transcripts. The neo+T4+ clones expressed a single full-length LTR-to-LTR transcript as detected by a T4 probe. The neo+T8+ clones, however, expressed both a full-length LTR-to-LTR transcript and an additional smaller transcript as detected by a T8 probe. This smaller transcript probably resulted from the utilization of cryptic signals which control 3' RNA processing. Furthermore, all of the neo+ clones expressed a transcript that initiated from the tk promoter, contained the neo gene, and used polyadenylation signals provided by the 3' LTR. Thus, the pMV-7 vector is capable of high-efficiency transfer and high-frequency expression of the cDNA-encoded protein.

Animals↗