Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “expression efficiency”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,603 records · Page 89Linked to original sources
PubMed1990

Temperature sensitive synthesis of transfer RNAs in vivo in Saccharomyces cerevisiae.

Dictyostelium discoideum tRNA genes can be expressed efficiently in vivo in yeast, and transcription products are processed to mature tRNAs. However, primary transcripts of a variant tRNA(Val)(UAC) gene are processing deficient under standard growth conditions (30 degrees C), due to a slightly altered 5' flanking region. A stable extended amino acid acceptor stem, which seems to be required to compensate a G5-G68 mismatch, cannot form. This mismatch destabilizes secondary and probably tertiary structures to such an extent that recognition of processing enzyme(s) under normal conditions (30 degrees C) is impaired. Growing yeast cells at reduced temperature (22 degrees C) can phenotypically complement the processing defect. This observation provides a new concept for the temperature dependent expression of protein coding genes which carry a nonsense codon. Translation of corresponding messages can be controlled by products of a temperature sensitive su-tRNA gene. We successfully tested this concept with two amber suppressors derived from a tRNA(Glu)(UUC) gene from D. discoideum. One of the variant tRNA genes codes for a product with a destabilized amino acid acceptor stem. Primary transcripts of this particular su-tRNA(Glu)(CUA) gene are processed only at reduced growth temperatures and consequently function as temperature sensitive suppressors only under these conditions.

Base Sequence↗
PubMed2006

Expression and secretion of Bifidobacterium adolescentis amylase by Bifidobacterium longum.

Bifidobacterium adolescentis Int-57 (INT57), isolated from human feces, secretes an amylase. We have shot-gun cloned, sequence analyzed and expressed the gene encoding this amylase in B. longum. The sequenced 2477 bp fragment was homologous to other extracellular amylases. The encoded protein was predicted to be composed of 595 amino acids with a molecular weight of 64 kDa, and was designated AmyB. Highly conserved amylase domains were found in AmyB. The signal sequence and cleavage site was predicted by sequence analysis. AmyB was subcloned into pBES2, a novel E. coli-Bifidobacterium shuttle vector, to construct pYBamy59. Subsequently, B. longum, with no apparent amylase activity, was transformed with pYBamy59. More than 90% of the amylase activity was detected in the culture broth. This approach may open the way for the development of more efficient expression and secretion systems for Bifidobacterium.

Amino Acid Sequence↗
PubMed2000

Application of DNA vaccine technology to aquaculture.

The aquaculture industry needs to augment its global production and efficiency to meet the increasing consumer needs for fish and shellfish products. Unfortunately, infectious diseases have been a major impediment to the development and profitability of fish farms. While vaccines offer the most efficient way to control infectious pathogens, current products have only been successful against some diseases. These are mostly bacterial, and there are still several important diseases, mainly of viral and parasitic origin, for which no prophylactic treatment exists. DNA vaccines, compared to traditional antigen vaccines, have several practical and immunological advantages that make them very attractive for the aquaculture industry. The early success of DNA vaccines in animal models was very encouraging, but fish are unique in many aspects, and findings with other classes of vertebrate, namely mammals and birds, do not necessarily apply to aquatic animals. However, more recent studies with reporter genes showed that fish cells efficiently express foreign proteins encoded by eukaryotic expression vectors. A piscine-specific backbone vector might eventually improve immune responses to DNA vaccines, but there is already strong direct evidence for the induction of protective immunity with currently available plasmids. Immune responses to plasmid DNA injected intramuscularly (IM) into fish are characterized by the production of antibodies, which have been shown to be neutralizing in two different viral disease models. There is also indirect evidence suggesting the induction of cell-mediated immunity. Despite this evidence, immune responses to DNA vaccines have only been poorly characterized in fish because of the limited knowledge of the piscine immune system, and the small number of studies on the subject. Apart from optimizing the efficiency of DNA vaccines, other important issues, such as safety and production cost will be determinants for the potential application of this technology in commercial fish farms. Alternative methods of administration will also have to be developed for small fish and low-valued species, for which IM injection is not practical and/or cost effective.

Administration, Oral↗
PubMed1990

Escherichia coli 4.5S RNA gene function can be complemented by heterologous bacterial RNA genes.

The essential 4.5S RNA gene of Escherichia coli can be complemented by 4.5S RNA-like genes from three other eubacteria, including both gram-positive and gram-negative organisms. Two of the genes encode RNAs similar in size to the E. coli species; the third, from Bacillus subtilis, specifies an RNA more than twice as large. The heterologous genes are expressed efficiently in E. coli, and the product RNAs resemble those produced by cognate cells. We conclude that the heterologous RNAs can replace E. coli 4.5S RNA and that the essential function of 4.5S RNA is evolutionarily conserved. A consensus structure is presented for the functionally related 4.5S RNA homologs.

Base Sequence↗
PubMed1982

Expression and excretion of human fibroblast beta 1 interferon in monkey cells after transfection with a recombinant SV40 plasmid vector.

We have constructed a eukaryotic expression vector designed to express a gene under late SV40 transcriptional control. From this chimeric plasmic-SV40 vector, virtually all the sequences which code for the major capsid protein VP1 have been deleted and instead, the human fibroblast interferon beta 1 cDNA gene has been inserted. After transfection of monkey cells with this recombinant, substantial quantities of human beta 1 interferon (up to 2 x 10(-4) IU/ml) were excreted in the culture medium. Transfection of nonpermissive mouse L cells or rat cells yielded virtually undetectable quantities of human beta 1 interferon (5 x 10(3) to 10(4) times less than that in monkey cells). The recombinant SV40 vector may serve as a model vehicle for the efficient expression of other eukaryotic genes and might also be used as a direct screening vector for cloning of eukaryotic or prokaryotic cDNA genes.

DNA, Recombinant↗
PubMed2003

Construction of vectors for inducible gene expression in Lactobacillus sakei and L plantarum.

We have constructed vectors for inducible expression of genes in Lactobacillus sakei and Lactobacillus plantarum. The key elements of these vectors are a regulatable promoter involved in the production of the bacteriocins sakacin A and sakacin P and the genes encoding the cognate histidine protein kinase and response regulator that are necessary to activate this promoter upon induction by a peptide pheromone. The vectors are built up of cassettes that permit easy exchange of all parts through restriction enzyme digestion and ligation. Using beta-glucuronidase as a reporter enzyme, variants of these vectors were compared with each other, and with a corresponding system based on genes involved in the production of nisin. Several of the new vectors permitted tightly controlled and efficient expression of beta-glucuronidase in both L. sakei and L. plantarum.

Gene Expression Regulation, Bacterial↗
PubMed2004

[Carboxyl terminus truncated HPV58 virus L1 protein expressed with baculovirus system and its bioactivity].

To prepare carboxyl terminus truncated human papillomavirus type 58L1 protein, and study on its in vitro bioactivity. PCR was used to amplify carboxyl terminus truncated HPV 58L1 gene, the product was inserted into the PUCMT cloning vector, preparing recombinant PFastBacHTb containing carboxyl terminus truncated HPV58L1 gene. Further more, the recombinant plasmid PfastbacHTb was used to transform DH10Bac cells, constructing recombinant Baculovirus, then the recombinant virus was successfully used to infect Sf-9 insect cells. After incubating at 27 degrees C for 72 hours, the infected cells were collected and total cellular proteins were extracted. The target protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The interested protein was purified by ProBond purification system. The purified interested protein was identified to self-assemble into VLPs by Transmission electron microscope, and induce murine erythrocyte hemagglutination, indicating that the given proteins had the conformation of VLPs, collecting, HPV58L1 proteins with carboxyl terminus truncation could be efficiently expressed in baculovirus Sf-9 cells expression system, it has identical in vitro bioactivity to the wild type HPV58L1, The present study is fundmental for preparing HPV58L1 prophylactic vaccine.

Animals↗
PubMed2001

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis).

A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3' region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5' and 3' untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence of H. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.

Amino Acid Sequence↗
PubMed1990

A novel upstream element compensates for an ineffectual octamer motif in an immunoglobulin V kappa promoter.

The octamer (or dc/cd) motif is considered to be a critical component of all immunoglobulin (Ig) promoters. Although the sequence of this motif is highly conserved among most Ig promoters, there are some notable examples in which efficiently expressed Ig genes contain divergent octamers with base substitutions that are demonstrably deleterious when tested with heterologous proximal promoter elements. To elucidate the mechanisms that enable these naturally occurring Ig genes to cope with divergent octamers, we analyzed two such promoters with regard to their ability to interact with relevant transcription factors. We found that the divergent octamer in the kappa O germline promoter strongly binds both Oct-1 and Oct-2 factors, presumably because of compensatory contributions by flanking DNA sequences. A more surprising result was obtained with the V kappa 19 promoter. In this case, the divergent octamer is a very weak Oct factor binding site and, without help from another upstream element, is inadequate for efficient promoter function. This additional element, termed kappa Y because of its high pyrimidine content (CTTCCTTA), serves as a binding site for a novel lymphoid-specific factor. When the divergent V kappa 19 octamer was converted to a strong Oct factor binding site by a single point mutation, the need for kappa Y was obviated. Interestingly, VH promoters that contain the same divergent octamer also contain an upstream element that is very similar to kappa Y.

Animals↗
PubMed1996

Characterization of the VIP receptor from SUP T1 lymphoblasts.

The SUP T1 lymphoblasts express an original subtype of VIP receptors characterized by a high affinity for the VIP analogue from lizard venom named helodermin, a preference for the neuropeptide PACAP-38 over PACAP-27 and VIP, and an extremely low affinity for secretin. The molecular cloning of that receptor revealed its identity with the VIP2 receptor subtype first cloned in rat and mouse tissues. The access to selective probes permits the detection of the mRNA coding for the VIP2 receptor by Northern blot, reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. These highly selective and sensitive techniques identify the cell types that are equipped to synthesize the receptor but do not prove that the receptor is indeed efficiently expressed at the cell surface. VIP2 mRNA was detected in selected areas of the brain different from that expressing the classical VIP1 receptor, in pituitary, in pineal, in pancreatic islets, in testes and ovary. It was also detected in the stomach, in the thymus and in spleen and in T lymphoblastic cell lines. A systematic screening of the immunocompetent cells must still be performed.

Amino Acid Sequence↗
PubMed1992

Recombinant human secretory phospholipase A2: purification and characterization of the enzyme for active site studies.

A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.

Animals↗
PubMed2004

Carboxyl terminus truncated human papillomavirus type 58 L1 protein maintains its bioactivity and ability to form virus-like particles.

To prepare carboxyl terminus truncated human papillomavirus type 58 L1 (HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1 protein, and pFastBac-Htb containing HPV58L1 gene sequence of carboxyl terminus truncation was generated. Then Sf-9 cells were infected with recombinant baculovirus. After being cultured, the post-infected cells expressing--HPV58L1 protein were harvested and analyzed by SDS-PAGE and Western blot. The ProBond purification system was used for protein purification. The bio-activity of purified protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was examined with transmission electron microscope. Our results showed that the recombinant baculovirus was generated and the Sf-9 cells was infected with the recombinant baculovirus, and after collecting, total cellular proteins were extracted. Truncated HPV58L1 protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The purified L1 proteins under native condition could cause mouse erythrocytes to agglutinate and form VLP. It is concluded that HPV58L1 protein with carboxyl terminus truncation could be efficiently expressed. In baculovirus Sf-9 cells expression system, the purified protein could self-assemble into virions in vitro, and induce agglutination of mouse erythrocytes, indicating that carboxyl terminus truncation does not interfere with the bioactivity of HPV58L1 protein.

Baculoviridae↗
PubMed1988

Integration host factor interacts with the DNA replication enhancer of filamentous phage f1.

We present data which show that the Escherichia coli integration host factor (IHF) is an activator of phage f1 DNA replication. Phage f1 poorly infects bacterial strains lacking IHF because IHF is required for efficient expression of F-pili, the receptor for f1 phage. However, when F- strains are transfected with f1 DNA the phage replicates in IHF mutants (himA, himD, or himA himD) at a rate of only 3% of that in wild-type bacteria. A plasmid dependent on the f1 replicon fails to transform IHF mutants. By gel retardation analysis, we show that IHF specifically binds to the origin of replication. DNase I "footprinting" experiments demonstrate that IHF binds to multiple sites within the replication enhancer sequence, a cis-acting, A + T-rich sequence that potentiates f1 DNA replication. Moreover, the effect of IHF mutation on f1 growth is suppressed by initiator protein (f1 gene II) mutations that restore efficient replication from origins that lack a functional replication enhancer sequence. This genetic evidence supports the conclusion that the replication enhancer sequence is the site of action of IHF.

Bacterial Proteins↗
PubMed2005

Molecular cloning of giant panda pituitary prolactin cDNA and its expression in Escherichia coli.

cDNA encoding pituitary (PRL) of giant panda was obtained using RT-PCR and expressed in E. coli. The results revealed that panda PRL cDNA encodes a precursor protein of 229 amino acids including a putative signal peptide of 30 amino acids and a mature protein of 199 residues with one potential N-glycosylation site. Sequence comparison indicated that panda PRL shares a high degree of identity to other known PRL sequences ranging from 98% with mink PRL to about 50% with rodent PRL. Six cysteine residues and 29 conserved residues distributed in four domains (PD1, PD2, PD3, and PD4) of PRL were observed. through multiple sequence alignment. Fourteen key residues of binding sites 1 and 2 involved in receptor binding are conserved in panda PRL. GST fused recombinant panda PRL protein was efficiently expressed with the form of insoluble inclusion bodies in E. coli BL21 transformed with a pGEX-4T-1 expression vector containing the DNA sequence encoding mature panda PRL. Western blot analysis indicated that GST-panda PRL recombinant protein could be recognized by antibody against human PRL. Our results would contribute to further elucidating the structural and functional characteristics of pituitary PRL and provide a basis for the production of recombinant panda prolactin for future use in the breeding of giant panda.

Amino Acid Sequence↗
PubMed1992

Integration host factor is required for positive regulation of the tdc operon of Escherichia coli.

A 14-bp segment in the promoter region of the tdcABC operon of Escherichia coli shows sequence identity with the consensus binding site for the E. coli integration host factor (IHF). In an himA (IHF-deficient) strain, expression of beta-galactosidase from a tdcB'-'lacZ protein fusion plasmid was about 10% of that seen with an isogenic himA+ strain. Threonine dehydratase activity from the chromosomal tdcB gene in the himA mutant was also about 10% of the wild-type enzyme level. Two different mutations introduced into the putative IHF-binding site in the fusion plasmid greatly reduced the plasmid-coded beta-galactosidase activity in cells containing IHF. In vitro gel retardation and DNase I footprinting analyses showed binding of purified IHF to the wild-type but not to the mutant promoter. IHF protected a 31-bp region between -118 and -88 encompassing the conserved IHF consensus sequence. These results suggest that efficient expression of the tdc operon in vivo requires a functional IHF and an IHF-binding site in the tdc promoter.

Amino Acid Sequence↗
PubMed2000

[Study on expression and resistance of the double drug resistance genes transduced into human umbilical cord blood CD34+ cells mediated by bicistronic retroviral vector].

To explore whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH1) and multidrug resistance gene (MDR1) increase resistance to 4-Hyaroxycyclophosphophamide (4-HC) and P-Glycoprotein Effluxed Drugs, a bicistronic Retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed. The vector was transduced into the packaging cell lines GP + E86 and PA317 by LipofectAMINE. Using the medium containing VCR and 4-HC for cloning selection and pingponging supernatant infection between ecotropic producer clone and amphotropic producer clone, we obtained high titer amphotropic PA317 producer clone with the highest titer up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfectced repeatedly with supernatant of retrovirus containing human ALDH1 and MDR1cDNA under stimulation of hemopoietie growth factors. PCR, RT-PCR, Southern blot, Northern blot, FACS and MTT method analyses show that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4- to 7.2-folds stronger resistance to cyclophospsphamede and P-Glycoprotein Effluxes drug in comparison with the nontransduced cells. This study provided a foundation for the application of combination chemotherapy in tumor clinical trial.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Functional expression of rat peroxisomal acyl-CoA oxidase in Spodoptera frugiperda cells.

A cDNA coding the 661-residue rat peroxisomal fatty acyl-CoA oxidase (ACOX) has been constructed and expressed as catalytically active protein in Spodoptera frugiperda (Sf9) insect cells using baculovirus expression system. Recombinant viral clones were purified and the expressed protein was identified by immunoblotting and catalytic activity. In the rat liver, ACOX consists of three polypeptide components A, B and C, with relative molecular mass of 72 kDa, 51 kDa and 21 kDa, respectively. In Sf9 insect cells infected with the recombinant virus, ACOX protein (component A) was expressed up to 15% of the total cellular protein. Immunoblot analysis demonstrated that besides component A, components B and C were also present in Sf9 cells, suggesting that these components are derived from component A by post-translational proteolytic cleavage. However, unlike in rat liver, baculovirus generated ACOX in insect cells had reduced amounts of components B and C with an estimated molar ratio of A, B, C of 5:1:1 in Sf9 cells vs 1:5:5 in rat liver. By immunofluorescence and immunocytochemical methods the overexpressed recombinant ACOX was identified both in the cytoplasm and the nucleus of Sf9 cells. Polyclonal antibodies raised against recombinant ACOX recognized rat liver ACOX on immunoblotting. Baculovirus Sf9 system provides high-efficiency expression of functional ACOX and can be used to express specific mutant and truncated ACOX cDNAs for further characterization.

Acyl-CoA Oxidase↗
PubMed1992

Cloning and inducible synthesis of poliovirus nonstructural proteins.

The poliovirus nonstructural protein-encoding genes have been cloned and expressed in Escherichia coli using the inducible system described by Studier and Moffat [J. Mol. Biol. 189 (1986) 113-130] and Studier [J. Mol. Biol. 219 (1991) 37-44]. The two genes encoding the poliovirus proteases, 2Apro and 3Cpro, were cloned together with their flanking regions in order to test the ability of the polyprotein precursors synthesized to cause proteolytic cleavage and generate mature forms. Both proteases were synthesized and showed activity upon induction in this system. Previously, it had not been possible to produce the three poliovirus nonstructural proteins, 2B, 2C and 3A, and some of their precursors, 2C3AB, 2C3A and 3AB, at high levels in E. coli cells. We report the cloning of their genes using PCR techniques and their efficient expression from pET vectors upon induction with IPTG (isopropyl-beta-D-thiogalactopyranoside). Moreover, some of these proteins, e.g., 3AB, 3A and 2B, are quite toxic for E. coli cells and lysed them upon production. Our results demonstrate the usefulness of this inducible system using the pET vectors to express these toxic poliovirus proteins.

3C Viral Proteases↗