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PubMed2006

[Expression of recombinant human hemangiopoietin and preparation of its polyclonal antibody].

AIM: To express the recombinant fusion protein of hemangiopoietin (HAPO) and prepare the rabbit-anti-human HAPO polyclonal antibody. METHODS: The sequence encoding HAPO was amplified by PCR and cloned into plasmid pET32c to construct recombinant prokaryotic expression system. The recombinant expression vectors were identified by enzyme digestion analysis and transformed into E. coli. The HAPO protein was purified by affinity chromatography. Rabbits were immunized with the HAPO protein, and the immune sera of rabbits were collected. Antibodies (IgG) obtained from the immune sera were purified. RESULTS: The purified HAPO protein was successfully obtained. The purified polyclonal antibody of rabbit-anti-human HAPO was also obtained from the immune sera of rabbits, and could response to human HAPO. CONCLUSION: A prokaryotic expression system of human HAPO has been prepared and the polyclonal antibody against HAPO has been prepared, which can be used to determine HAPO protein.

Animals↗

PubMed1997

Enhancement of recombinant glucoamylase expression by introducing yeast GAL7 mRNA termination sequence.

Glucoamylase gene (STA1) of Saccharomyces diastaticus was expressed in recombinant Saccharomyces cerevisiae systems. The yeast, GAL7 mRNA termination sequence, was introduced in the 3' noncoding region of the STA1 structural gene which was under the control of the SUC2 promoter and STA1 secretion signal sequence. This plasmid was named YEpSSG7 and was introduced into yeast S. cerevisiae MMY2 to construct recombinant S. cerevisiae MMY2SSG7. The GAL7 mRNA termination sequence enhanced the glucoamylase expression level by 3-5 times depending on the culture conditions compared to the result from the strain S. cerevisiae MMY2SUCSTA which did not contain the GAL7 mRNA termination sequence. Such an enhancement was not due to plasmid stability or plasmid copy number effects. Such an enhancement was primarily due to the fact that GAL7 mRNA termination sequence stabilized the STA1 mRNA 3' end.

Deoxyribonuclease EcoRI↗

PubMed1999

The effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein D of bovine herpesvirus type 1.

We investigated whether pre-existing adenovirus-specific immunity influenced the development of immunity to a foreign antigen expressed by recombinant adenovirus. Active adenovirus-specific immunity was induced in cotton rats by i.n. administration of wild type human adenovirus type 5 (HAd5) two weeks before immunisation with a HAd5 vector expressing the glycoprotein D (gD) of bovine herpesvirus type 1 (gD-dE3 recombinant adenovirus). Active adenovirus-specific immunity inhibited gD-specific immune responses, following either i.n. or gastrointestinal immunisation with gD-dE3. An inhibitory effect was present even if infection with HAd5 and immunisation with gD-dE3 were 13 weeks apart. Passive transfer of adenovirus specific antibodies to cotton rats one day before immunisation, however, did not significantly inhibit gD-specific immune responses induced by i.n. immunisation with gD-dE3. Repeated administration of an adenovirus vector, therefore, may have a limited ability to deliver antigen, while passive immunity to adenovirus may not interfere with the success of immunisation.

Adenoviruses, Human↗

PubMed1994

Expression of recombinant E2 and C proteins of rubella virus in insect cells.

We have constructed a recombinant baculovirus expressing the rubella virus E2 (42-45 KDa) and C (34 KDa) proteins. Sf9 cells infected with recombinant virus were able to synthesize and process the two proteins coded by a unique precursor gene. By immunoblot and immunoprecipitation analysis with polyclonal and monoclonal antibodies, a precursor polyprotein (66 KDa) and two other proteins migrating with an apparent molecular weight of 42 KDa and 36 KDa were recognized as E2 glycoprotein and C protein, respectively. The recombinant E2 protein appeared to be glycosylated since it was susceptible to tunicamycin. The results indicate that the RV polyprotein coding for E2 and C is expressed and proteolytically cleaved in insect cells. This baculovirus expression system provides a useful alternative approach for the production of rubella virus antigens and should allow the purification of large quantities of the RV proteins for further biochemical and immunological studies.

Animals↗

PubMed1996

Recombinant-baculovirus-expressed PB2 subunit of the influenza A virus RNA polymerase binds cap groups as an isolated subunit.

The influenza A virus RNA-dependent RNA polymerase catalyzes several reactions in transcription and replication of the genome RNA. The first step in viral mRNA synthesis is the recognition of the 5' end cap structure of host cell hnRNA and the cleavage of the RNA substrate between 10 and 14 nucleotides from the 5' end to generate capped primers for initiation of transcription of virus-specific mRNAs. This report describes the use of an in vitro UV crosslinking and protein renaturation assay to identify the polymerase subunits which interact with the 5' end cap structure of an artificial RNA substrate. Our results showed, for the first time, that purified polymerase subunit PB2 expressed by recombinant baculovirus in insect cells possessed cap-binding activity by itself after renaturation by Escherichia coli thioredoxin, whereas cleavage of the artificial capped substrate required the holoenzyme expressed in insect cells triply-infected with baculovirus containing all three polypeptide components, PB1, PB2, and PA. Purified polyclonal anti-PB2 IgG inhibited the binding activity; anti-PB1 and anti-PA IgGs did not.

Animals↗

PubMed1990

Increased antibody responses to human papillomavirus type 16 L1 protein expressed by recombinant vaccinia virus lacking serine protease inhibitor genes.

The L1 gene of human papillomavirus type 16 (HPV-16) driven by the vaccinia virus major late 4b gene promoter has been inserted into three different sites of the vaccinia virus genome. Insertion into the thymidine kinase (TK) gene was achieved by selection of TK- mutants in BUdR on TK- cells. Insertion into two vaccinia virus serine protease inhibitor (serpin) genes was achieved by co-insertion of the Escherichia coli xanthine guanine phosphoribosyltransferase gene linked to the vaccinia virus 7.5K promoter and selection of mycophenolic acid-resistant recombinant viruses. Each recombinant virus expressed a 57K L1 protein at similar levels and with similar kinetics. However, immunization of mice with these recombinant viruses induced different levels of antibody to the L1 protein. Viruses lacking serpin genes B13R and B24R induced significantly higher antibody levels than did viruses lacking the TK gene. The presence of functional B13R and B24R gene products is therefore somehow immunosuppressive at least for antibody responses to the L1 protein of HPV-16.

Animals↗

PubMed2004

Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin.

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.

Actins↗

PubMed1996

Construction of bovine herpesvirus-1 (BHV-1) recombinants which express pseudorabies virus (PRV) glycoproteins gB, gC, gD, and gE.

We have improved the method for constructing recombinants of bovine herpesvirus type-1 (BHV-1). Using this method, we constructed three recombinants in which the pseudorabies virus (PRV) thymidine kinase (tk) gene was inserted at three different sites in the unique short region of BHV-1. These three sites are located in the open reading frame of gE, gG and gI genes. Previously, two sites (tk and gC) had been used to insert foreign DNA fragments to BHV-1 genome. Therefore we now have 5 sites in BHV-1 where DNA can be inserted. The gB, gC, gD, gE and gI genes of PRV were successfully inserted at the tk or the gC gene of BHV-1 genome and Western blot analyses confirmed that the recombinants express PRV gB, gC, gD and gE. Anti-PRV gB and gC antibodies as well as anti-PRV polyclonal serum neutralized BHV-1 recombinants which express PRV gB and gC. The latter was neutralized more strongly. However, anti-gD monoclonal antibody and anti-PRV polyclonal serum failed to neutralize gD-expressing recombinants. This suggests that PRV gC and some gB are integrated into the viral envelope of the recombinants, but very little gD is present in the viral envelope.

Animals↗

PubMed1994

Biochemical characterization of Drosophila melanogaster acetylcholinesterase expressed by recombinant baculoviruses.

Recombinant baculoviruses expressing full length and 3' truncated forms of c-DNA encoding the Drosophila melanogaster acetylcholinesterase (AChE) were constructed. Biochemical analyses showed that full length recombinant protein was enzymatically active and anchored to the cell membrane via a glycolipidic residue. DTT treatment dissociated the native form into monomers migrating as did the corresponding form of AChE extracted from drosophila heads. Finally, DFP labelling demonstrated that the specific proteolytic cleavage leading to the formation of 55 and 16 kDa subunits occurred in Sf9 cells. In contrast with the full-length enzyme, C-terminal-truncated forms were highly secreted, confirming the prominent role of the C-terminal hydrophobic peptide for the addition of the glycolipidic residue. Accumulation of inactive precursor was observed when recombinant proteins were overproduced using an improved baculovirus, suggesting a saturation of insect cell machineries.

Acetylcholinesterase↗

PubMed1999

[Stable expression of recombinant inducible nitric oxide synthase in NG108-15 cells and its biological characterization].

The neuroblastoma x glioma NG108-15 cells were transfected with recombinant eukarytic expression plasmid pCMViNOS containing the full-length cDNA encoding inducible nitric oxide synthase (iNOS). A lot of G418-resistant clones were screened at 600 micrograms/ml of geneticin. In the 2# clone expressing iNOS gene, iNOS catalytic activity in the cytosol fraction displayed to have an increasing trend, accompanying with the accumulation of NO2- content in the supernantant of cultured cells and the intracellular cGMP concentration, which suggested that NO-cGMP signal pathway was mediated by the expression of iNOS gene and blocked by NG-nitro-L-arginine (L-NNA) and methylene blue (MB). Activity of iNOS was concentration-dependently inhibited by NOS inhibitors such as L-NNA and aminoguanidine. The result of measurement of NADPH diaphorase activity and immunocytochemical staining showed that localization of the function expression of iNOS protein mainly existed in the cytoplasm of NG108-15 cells transfected with pCMViNOS. Furthermore, the chromosomal integration, transcript and protein translation of foreign iNOS gene were identified by Southern hybridization, RT-PCR and Western blot, respectively. The results indicated that iNOS gene-transfected cells had mRNA transcription and specific protein expression at high level. Given the above results, the engineering cell line with stable expression of iNOS gene was successfully established. The new neuronal cell line may serve as a source of iNOS and provide a useful cell model for studying iNOS biological function and developing novel iNOS-selective inhibitors.

Animals↗

PubMed1988

Passive transfer of respiratory syncytial virus (RSV) antiserum suppresses the immune response to the RSV fusion (F) and large (G) glycoproteins expressed by recombinant vaccinia viruses.

In young infants who possess maternally derived respiratory syncytial virus (RSV) antibodies, the antibody response to RSV glycoproteins is relatively poor, despite extensive replication of RSV. In the present study, it was found that cotton rat RSV hyperimmune antiserum suppressed the antibody response to the RSV glycoproteins but not the response to vaccinia virus antigens when the antiserum was passively transferred to cotton rats prior to infection with vaccinia recombinant viruses expressing the RSV envelope glycoproteins. The cotton rats which had their immune responses suppressed by passively transferred antibodies were more susceptible to infection with RSV than were animals inoculated with control serum lacking RSV antibodies. Furthermore, many of the immunosuppressed animals infected with the vaccinia recombinant viruses developed RSV glycoprotein antibodies which had abnormally low neutralizing activities. Thus, preexisting serum RSV antibodies had dramatic quantitative and qualitative effects on the immune response to RSV glycoproteins, which may explain, in part, the poor RSV antibody response of young human infants to infection with RSV. Our observations also suggest that immunosuppression by preexisting, passively acquired RSV antibodies may constitute a major obstacle to RSV immunoprophylaxis during early infancy, when immunization is most needed.

Animals↗

PubMed1992

New vectors for high level expression of recombinant proteins in bacteria.

A system has been developed for synthesis and rapid purification of recombinant polypeptides expressed in frame with glutathione S-transferase (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). Expressed fusion proteins are purified from bacterial extracts by glutathione-agarose affinity chromatography. A thrombin protease cleavage site allowed for proteolysis of the fusion protein. We reported the construction of the vector pGEX-KG (K. Guan and J. E. Dixon, 1991, Anal. Biochem. 192, 262-267) which has a glycine-rich "kinker" immediately after the thrombin cleavage site. This kinker dramatically improved the thrombin cleavage efficiency of several fusion proteins. One potential drawback of expressing proteins in this vector is that, following proteolytic cleavage, unrelated amino acids from the vector remain at the amino terminus of the released protein. These extensions could affect enzymatic activity or protein structure. We have constructed two new vectors, pGEX-KT and pGEX-KN, which have the glycine kinker placed N-terminal to the thrombin cleavage site in order to minimize the unrelated amino acids associated with the cleaved protein. The change in location of the kinker had no effect on the increased thrombin cleavage efficiency. A strategy combining the kinker in the vector pGEX-KN with polymerase chain reaction has also been developed to express fusion proteins which when cleaved with thrombin released a protein having no amino terminal extensions of any kind.

Base Sequence↗

PubMed1992

Expression of recombinant myeloperoxidase using a baculovirus expression system.

Myeloperoxidase (MPO) is a glycosylated heme-containing enzyme present in the azurophilic granules of normal human polymorphonuclear neutrophils. This enzyme plays a major role in the microbicidal activity of the host defense system by catalyzing the formation of the potent oxidant, hypochlorous acid. Although the amino acid sequence of MPO has been deduced from the cDNA, the structural basis for the observed heterogeneity of this enzyme is not known. Furthermore, the nature of the prosthetic group and its mode of linkage to the apoprotein has not been determined. To address questions regarding the structural features of MPO, which arise during the complex posttranslational processing of this enzyme, we utilized a baculovirus system to express MPO in Sf9 insect cells. Two glycosylated, single-chain precursor species of MPO were observed: an 84 kDa species that was secreted and a 74 kDa species that was cell-associated. This is the first report of an expression system in which a cell-associated MPO precursor undergoes posttranslational proteolytic processing.

Animals↗

PubMed1996

Pharmacological analysis of dopamine stimulation of [35S]-GTP gamma S binding via human D2short and D2long dopamine receptors expressed in recombinant cells.

1. The activation of G-proteins by agonist-occupied D2 or D3 dopamine receptors in membranes from recombinant cells expressing the cloned receptors has been analysed by a [35S]-guanosine 5'-[gamma-thio] triphosphate ([35S]-GTP gamma S) binding assay. 2. The rate of [35S]-GTP gamma S binding was increased by dopamine in a dose-dependent manner in membranes from CHO cells stably expressing either the D2short or D2long dopamine receptor. 3. The dopamine-induced stimulation of [35S]-GTP gamma S binding could be inhibited by a range of antagonists. Affinities for antagonists derived from the inhibition of the dopamine stimulation of [35S]-GTP gamma S binding correlated very well with affinities derived from radioligand binding studies. 4. When the maximum [35S]-GTP gamma S binding responses stimulated by dopamine acting at different receptor subtypes were compared, there was a tendency for the stimulation via the D2short receptor to be greater than via the D2long receptor and for the stimulation via the D3 dopamine receptor to be less than for either D2 receptor. These differences in maximal response were also seen when the inhibitory effects of dopamine on adenylyl cyclase via the three receptor subtypes were compared. 5. The stimulation of [35S]-GTP gamma S binding by dopamine in membranes from recombinant cells therefore provides an excellent system for studying the molecular nature of agonism and the receptor/G-protein interactions for these receptors.

Adenylyl Cyclases↗

PubMed1999

Rat muscle fructose-1,6-bisphosphatase: cloning of the cDNA, expression of the recombinant enzyme, and expression analysis in different tissues.

The 1282 bp cDNA of an isoenzyme of fructose-1,6-bisphosphatase was cloned from rat muscle. It shows 70% positional identity to the cDNA of rat liver fructose-1,6-bisphosphatase and is clearly the product of a gene different from that coding for the liver enzyme. After cloning of the coding region of the rat muscle fructose-1,6-bisphosphatase cDNA in an expression vector, the recombinant enzyme could be detected in E. coli cell-free extracts by activity determination and Western blotting. Overexpressed fructose-1,6-bisphosphatase was found to be allosterically inhibited by AMP comparably to the enzyme isolated from rat muscle. Analysis of steady-state mRNA levels of various rat tissues with reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blotting revealed one or the two fructose-1,6-bisphosphatase isoenzyme mRNAs in most tissues tested with significant quantitative differences. Quantitative PCR using a homologous competitor showed that 1 microg of total RNA of rat muscle contains 1.7 x 10(6) molecules of rat muscle fructose-1,6-bisphosphatase mRNA. 3 x 10(4) copies of this message were found per microg total RNA of heart and kidney, respectively.

Amino Acid Sequence↗

PubMed1991

Recombinant rhizopuspepsinogen. Expression, purification, and activation properties of recombinant rhizopuspepsinogens.

A cDNA clone, which contained the complete rhizopuspepsin structure and the putative proregion, was placed in three different Escherichia coli expression vectors for the synthesis of rhizopuspepsinogen (Rpg). Recombinant Rpgs which were expressed in the cytosol of E. coli as inclusion bodies (cRpg and tRpg) were not active. After solubilization in 6 M urea and refolding by rapid dilution, both of these Rpgs were purified to homogeneity. The third zymogen, pRpg, which was secreted to the periplasmic space of E. coli with an omp leader, was fully active and also was purified. The expression level of pRpg was higher (over 40 mg/liter culture) than that of cRpg (about 1.5 mg/liter culture). Amino-terminal sequence analysis of the zymogens revealed that cRpg and pRpg contain 40 and 51 residues of prosequence, respectively. tRpg, which was expressed under the control of T7 promoter, was synthesized at 500 mg/liter culture and was purified at 50 mg/liter culture. This zymogen contained, in addition to 51 residues of proregion, 16 residues inherited from the expression vector construction. All of these Rpgs spontaneously converted to rhizopuspepsin in solutions of pH less than 5. Each of the conversions was associated with a change of molecular weight as monitored in sodium dodecyl sulfate-polyacrylamide electrophoresis. At least one intermediate of conversion was observed in the pH range of 2 to 3 for both the cRpg and pRpg zymogens. For pRpg and tRpg, kinetic data demonstrated that the Rpg to rhizopuspepsin conversion was accomplished by a first order, unimolecular reaction at pH 2. The first order kinetic constants in this pH at 15 degrees C were 1.1 and 2.4 min-1 for pRpg and tRpg, respectively. The activation rate decreased as pH was raised above pH 2. At pH greater than 3.0, rhizopuspepsin-catalyzed, second-order activation also takes place. Consequently, the recombinant Rpgs are activated by either of two cleavage mechanisms as is the case for pepsinogen. These results also support the hypothesis that Rpg is synthesized in Rhizopus chinensis as a zymogen. Rpg in the host fungus is probably activated by an acid environment of pH less than 5 in the secretory granules to become rhizopuspepsin before secretion.

Amino Acid Sequence↗

PubMed2002

A rapid screening method to monitor expression of recombinant proteins from various prokaryotic and eukaryotic expression systems using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

Rapid methods using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to monitor recombinant protein expression from various prokaryotic and eukaryotic cell culture systems were devised. Intracellular as well as secreted proteins from both induced and constitutive expression systems were measured and monitored from whole cells and growth media, thus providing an alternative to time-consuming traditional methods for screening and monitoring of protein expression. The methods described here involve minimal processing of samples and are therefore relevant to high-throughput screening applications.

Animals↗

PubMed1990

A dual-affinity gene fusion system to express small recombinant proteins in a soluble form: expression and characterization of protein A deletion mutants.

A novel gene fusion system to express and purify small recombinant proteins in Escherichia coli has been constructed. The concept allows for affinity purification of soluble gene products by sequential albumin- and Zn2(+)-affinity chromatography. The dual-affinity system is well suited for expression of unstable proteins as only full-length protein is obtained after purification and proteins gain proteolytic stability in the fusion protein. Here we show that the dual-affinity approach can be used for the expression of various unstable derivatives of a single IgG-binding domain based on staphylococcal protein A. Analysis of the proteolytic stabilities and the IgG-binding properties of the different mutant proteins suggest that the model for the structure of an IgG-binding domain must be re-evaluated.

Base Sequence↗