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Results for “Recombinant expression”

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PubMed2004

High-throughput screening for soluble recombinant expressed kinases in Escherichia coli and insect cells.

We have constructed a dual expression vector for the production of recombinant proteins in both Escherichia coli and insect cells. In this vector, the baculoviral polyhedrin promoter was positioned upstream of the bacteriophage T7 promoter and the lac operator. This vector, designated pBEV, was specifically designed to exploit the advantages that both hosts would provide. This vector also facilitates one-stop cloning, thereby simplifying the expression process for automation, and the development of a high-throughput method for protein expression. Utilizing the multi-system vector pBEV, a high-throughput process was developed with expression in deep-well blocks and purification in micro-titer plates enabling the identification of expression and solubility in both E. coli and insect cells. In this study, using pBEV, we have successfully expressed and purified multiple human kinases produced in E. coli and insect cells. Our results validate expression screening as a strategy to rapidly triage proteins identifying the optimum expression system and conditions for production.

Animals↗

PubMed1995

Formation of 4,4'-methylene-bis(2-chloroaniline)-DNA adducts in yeast expressing recombinant cytochrome P450s.

N-Oxidation of 4,4'-methylene-bis(2-chloroaniline) (MBOCA) may lead to formation of DNA adducts. To determine if cytochrome P450s are involved in the formation of MBOCA derived-DNA adducts, yeast strains expressing rodent P450s were exposed to MBOCA, and 32P-postlabelling of nucleotides from yeast genomic DNA was done. Chromatographic analysis on PEI cellulose showed that, upon exposure to MBOCA for 1 h, nine DNA adducts were formed in yeast expressing phenobarbital-inducible rabbit P450 2B5. With a 4-h-exposure, all adducts increased in parallel. In cell-free experiments, the incubation of MBOCA with phenobarbital-induced rat microsomal fraction followed by incubation with thymus DNA, led to the formation of more than ten DNA adducts. When yeast expressing 3-methylcholanthrene-inducible rat P450 1A1 was exposed to MBOCA, one major and two minor adducts were formed. No adducts were detected in control yeast. These results show that recombinant rabbit P450 2B5 exhibits a potential activation of MBOCA and that rat P450 1A1 has some effect. The use of yeast expressing recombinant P450s and the technique of 32P-postlabelling facilitates a simple search for chemicals with carcinogenic potential.

Cytochrome P-450 Enzyme System↗

PubMed2002

Evaluation of the immunocontraceptive potential of Escherichia coli-expressed recombinant dog ZP2 and ZP3 in a homologous animal model.

Dog zona pellucida glycoprotein 2 (dZP2), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned and expressed as a polyhistidine fusion protein in Escherichia coli to evaluate the immunocontraceptive efficacy of ZP glycoproteins. The recombinant dZP2 (rec-dZP2) revealed a 70 kDa band corresponding to the full length transcript, as well as several low molecular mass fragments in western blot analysis. In addition to rec-dZP2, E. coli expressed recombinant dog ZP glycoprotein 3 (rec-dZP3), which has also been evaluated for its efficacy to block fertility in a homologous system. Three groups of female dogs (n = 4 per group) were immunized with rec-dZP2 conjugated to diphtheria toxoid (rec-dZP2-DT), rec-dZP3 conjugated to DT (rec-dZP3-DT) and DT alone. Immunization of female dogs with rec-dZP2-DT and rec-dZP3-DT led to generation of antibodies against the respective ZP proteins as well as to DT. Subsequent to mating, the four female dogs immunized with rec-dZP2-DT all conceived, which is indicative of failure of the anti-rec-dZP2 antibodies to block fertility. In the group of dogs immunized with rec-dZP3-DT, three of four animals did not conceive when mated with males of proven fertility. The block in fertility was associated with anti-dZP3 antibody titres. Ovarian histopathology revealed that the block in fertility in the group immunized with rec-dZP3-DT is probably manifested by inhibition in the development of follicles and is due to atretic changes in the zona pellucida. These results, although preliminary, indicate that immunization with dZP3 may be a feasible proposition to control dog populations provided that adequate antibody titres are achieved.

Animals↗

PubMed2004

E. coli-expressed recombinant norovirus capsid proteins maintain authentic antigenicity and receptor binding capability.

The baculovirus expression system has been widely used to produce the capsid proteins of Norovirus (NV) and the proteins form virus-like particles (VLPs) that are useful in many studies, such as immunology, diagnosis, and host-receptor interaction. We report here the application of the E. coli expression system in the production of recombinant NV capsid proteins. In a direct comparison of a previous well-characterized NV strain (VA387), we have demonstrated that the E. coli-expressed capsid proteins maintain the same antigenicity and receptor binding specificity as that of the baculovirus-expressed capsid, although the E. coli-expressed VA387 proteins did not form VLPs. Using the E. coli-expression system, we characterized the receptor-binding patterns of three additional NV strains (OIF1998, Parris Island and VA115), in which OIF1998 binds to HBGA of nonsecretors but did not bind or binds weakly to the HBGA of secretors, as seen in strain VA207. Parris Island binds to HBGA of types A and B but not type O secretors and nonsecretors. VA115 did not show specific binding to any A, B, O secretor nor nonsecretor, which is also observed when the capsid protein of this strain was expressed in baculovirus. Our data indicate that VLP formation is not required for receptor binding, and that the bacteria expression system offers a simple alternative for large production of NV capsid protein for various research purposes, particularly for strains generating low yields in the insect cells.

Antigens, Viral↗

PubMed1995

Human monoclonal antibodies to cytomegalovirus. Characterization and recombinant expression of a glycoprotein-B-specific antibody.

Human monoclonal antibodies (mAb) to human cytomegalovirus (HCMV) were established from spleen cells of a HCMV-positive donor. The antibodies (gamma 3, lambda) secreted from a stable heterohybridoma cell line were further characterized by immunoprecipitation and immune-fluorescence microscopy using HCMV infected cells and recombinant cell lines expressing HCMV glycoprotein B. The antibody reacted with the entire glycoprotein B or the extracellular domain expressed as glycoprotein-B--beta-galactosidase fusion protein in the native state, but the antibody was not neutralizing HCMV. Denatured and reduced forms of glycoprotein B were not recognized by this antibody, however, native glycoprotein B on the surface of infected cells was detected efficiently. The genes encoding the Fab part of the antibody were cloned and expressed in Escherichia coli. Recombinant Fab fragments specifically binding the extracellular domain of glycoprotein B could easily be isolated from the periplasmic space. Recombinant antibodies provide the opportunity to modify effector functions and to add tags to diagnostic antibodies for more efficient detection of CMV-infected cells.

Amino Acid Sequence↗

PubMed1997

Fermentation of biomass-derived glucuronic acid by pet expressing recombinants of E. coli B.

The economics of large-scale production of fuel ethanol from biomass and wastes requires the efficient utilization of all the sugars derived from the hydrolysis of the heteropolymeric hemicellulose component of lignocellulosic feedstocks. Glucuronic and 4-O-methyl-glucuronic acids are major side chains in xylans of the grasses and hardwoods that have been targeted as potential feedstocks for the production of cellulosic ethanol. The amount of these acids is similar to that of arabinose, which is now being viewed as another potential substrate in the production of biomass-derived ethanol. This study compared the end-product distribution associated with the fermentation of D-glucose (Glc) and D-glucuronic acid (GlcUA) (as sole carbon and energy sources) by Escherichia coli B (ATCC 11303) and two different ethanologenic recombinants--a strain in which pet expression was via a multicopy plasmid (pLOI297) and a chromosomally integrated construct, strain KO11. pH-stat batch fermentations were conducted using a modified LB medium with 2% (w/v) Glc or GlcUA with the set-point for pH control at either 6.3 or 7.0. The nontransformed host culture produced only lactic acid from glucose, but fermentation of GlcUA yielded a mixture of ethanol, acetic, and lactic acids, with acetic acid being the predominant end-product. The ethanol yield associated with GlcUA fermentation by both recombinants was similar, but acetic acid was a significant by-product. Increasing the pH from 6.3 to 7.0 increased the rate of glucuronate fermentation, but it also decreased the ethanol mass yield from 0.22 to 0.19 g/g primarily because of an increase in acetic acid production. In all fermentations there was good closure of the carbon mass balance, the exception being the recombinant bearing plasmid pLOI297 that produced an unidentified product from GlcUA. The metabolism of GlcUA by this metabolically engineered construct remains unresolved. The results offered insights into metabolic fluxes and the regulation of pyruvate catabolism in the wild-type and engineered strains. End-product distribution for metabolism of glucuronic acid by the nontransformed, wild-type E. coli B and recombinant strain KO11 suggests that the enzyme pyruvate-formate lyase is not solely responsible for the production of acetylCoA from pyruvate and that derepressed pyruvate dehydrogenase may play a significant role in the metabolism of GlcUA.

Acetyltransferases↗

PubMed1991

A vaccinia virus double recombinant expressing the F and H genes of rinderpest virus protects cattle against rinderpest and causes no pock lesions.

Rinderpest is a highly contagious viral disease of ruminants with greater than 95% morbidity and mortality. We have constructed an infectious vaccinia virus recombinant that expresses both the fusion (F) gene and the hemagglutinin (H) gene of rinderpest virus. The Wyeth strain of vaccinia virus was used for the construction of the recombinant. Cattle vaccinated with the recombinant virus were 100% protected from challenge inoculation with greater than 1000 times the lethal dose of rinderpest virus. No transmission of recombinant vaccinia virus from vaccinated animals to contact animals was observed. The lyophilized form of vaccinia virus is thermostable and allows circumvention of the logistical problems associated with the distribution and administration of vaccines in the arid and hot regions of Asia and Africa. The insertional inactivation of both the thymidine kinase and the hemagglutinin genes of vaccinia virus led to increased attenuation of the virus; this was manifested by the lack of detectable pock lesions in vaccinated animals. This approach may have wide application in the development of safe and efficacious recombinant vaccines for humans and animals. This becomes quite relevant with the concern of the use of vaccinia virus in a population with high incidence of the human immunodeficiency virus.

Animals↗

PubMed2005

Generation of eGFP expressing recombinant Zaire ebolavirus for analysis of early pathogenesis events and high-throughput antiviral drug screening.

Zaire ebolavirus causes large outbreaks of severe and usually fatal hemorrhagic disease in humans for which there is no effective treatment or cure. To facilitate examination of early critical events in viral pathogenesis and to identify antiviral compounds, a recombinant Zaire ebolavirus was engineered to express a foreign protein, eGFP, to provide a rapid and sensitive means to monitor virus replication in infected cells. This genetically engineered virus represents the first insertion of a foreign gene into ebolavirus. We show that Ebola-eGFP virus (EboZ-eGFP) infects known early targets of human infections and serves as an ideal model to screen antiviral compounds in less time than any previously published assay.

Animals↗

PubMed2004

Serological identification of endothelial antigens predominantly recognized in Kawasaki disease patients by recombinant expression cloning.

We showed IgG immune response against endothelial antigens in sera obtained from convalescent Kawasaki disease (KD) patients after recovery from the disease during the follow-up period using serological analysis of recombinant cDNA expression library (SEREX) methodology. We identified 46 antigens represented by 69 clones by immunoscreening of a cDNA expression library from tumor necrosis factor-alpha (TNFalpha) treated human umbilical vein endothelial cells (HUVEC) with sera from 4 KD patients. They included ubiquitin pathway proteins, transcriptional factors, signal transduction molecules, metabolic enzymes, cytoskeletal proteins, an adhesion molecule, and a cell cycle protein. By serological survey using phage plaque assay, sera from 5 non-KD patients were rarely reactive with the antigens. Among the antigens, tropomyosin was most frequently isolated (18 of 69 clones). Seventeen of the 18 clones were identified using KD3 serum. The second most frequently isolated antigen was also a cytoskeletal protein, called Tplastin (3 of 69 clones).

Antibodies↗

PubMed1997

Pseudorabies virus recombinants expressing functional virulence determinants gE and gI from bovine herpesvirus 1.1.

In the Alphaherpesvirinae subfamily, the gE and gI genes are conserved and encode membrane glycoproteins required for efficient pathogenesis (virulence). The molecular mechanism(s) responsible is not well understood, but the existence of similar phenotypes of gE and gI mutations in diverse Alphaherpesvirinae implies conservation of function(s). In this report, we describe construction of pseudorabies virus (PRV) recombinants that efficiently express the bovine herpesvirus 1 (BHV-1) membrane proteins gI and gE at the PRV gG locus. Each BHV-1 gene was cloned in a PRV mutant lacking both the PRV gI and gE coding sequences. All recombinant viruses expressed the BHV-1 proteins at levels similar to or greater than that observed after infection with parental BHV-1, and there were no observable differences in processing or ability to form gE-gI oligomers. The important observation resulting from this report is that the BHV-1 gE and gI proteins functioned together to complement the virulence defect of PRV lacking its own gE and gI genes in a rodent model, despite being derived from a highly restricted host range virus with a different pathogenic profile.

Animals↗

PubMed1999

Effect of virulence on immunogenicity of single and double vaccinia virus recombinants expressing differently immunogenic antigens: antibody-response inhibition induced by immunization with a mixture of recombinants differing in virulence.

It has been shown recently that the residual virulence of vaccinia virus (VV) is an important factor that influences the outcome of immunization with VV recombinants. This study focused on the correlation of the residual virulence of several VV recombinants with antibody responses against the strongly immunogenic extrinsic glycoprotein E of varicella-zoster virus and the weakly immunogenic extrinsic protein preS2-S of hepatitis B virus and against VV proteins, with mice used as a model organism. Furthermore, the effects of mixing different recombinants on the antibody response were studied. The results obtained indicated that: (i) the antibody response depended on the residual virulence of the recombinants, more so in the case of the weakly immunogenic protein; (ii) the residual virulence, the growth rate of the VV recombinants in extraneural tissues and the immunogenicity were associated features; (iii) immunization with mixtures of two differently virulent recombinants or with unequal amounts of two similarly virulent recombinants sometimes led to the suppression of antibody response. The appearance of this suppression was dependent on three factors: the residual virulence of the recombinants, the immunogenicity of the extrinsic proteins and the ratio of the recombinants in the mixtures. Thus, the data obtained demonstrate that there are various limitations to the use of replicating VV recombinants for immunization purposes.

Animals↗

PubMed1991

Vaccinia recombinants expressing secreted and transmembrane forms of breast cancer-associated epithelial tumour antigen (ETA).

Monoclonal antibody H23 identifies a polymorphic epithelial tumour antigen (ETA) that is aberrantly expressed in breast cancer and which may afford a target for active immunotherapy. We recently reported the cloning of H23-ETA genomic and cDNA clones. H23-ETA contains a multiple internal tandem repetition of a 20 amino acid motif and sequence analysis predicted two mRNA species encoding different ETA proteins, one harbouring a C-terminal potentially transmembrane hydrophobic zone (T) and a second form (S) that lacks this zone. We report that both RNA species can be detected in breast cancer cells. To further characterize the encoded proteins we have constructed vaccinia virus recombinants, VV-ETA-S and VV-ETA-T, separately expressing the alternative forms. Despite selective loss of internal tandem repeat elements during propagation of recombinant vaccinia, the encoded polypeptides were efficiently recognized by H23 monoclonal antibody. Immunoprecipitation revealed that ETA encoded by the S recombinant was secreted into the culture medium whereas the T form remained tethered at the cell surface. Both forms were readily detected in infected cells by immunofluorescence. Abnormal mobility of the T polypeptide indicated post-translational cleavage that may permit the extracellular domain of the T-polypeptide to be shed from the cell surface. Further, fluorescence-activated cell sorting analysis shows that the S form of the polypeptide is also partly present at the cell surface. Vaccinia recombinants expressing ETA may be of utility in the active immunotherapy of breast cancer.

Antigens, Neoplasm↗

PubMed2004

Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli.

The production of correctly folded protein in Escherichia coli is often challenging because of aggregation of the overexpressed protein into inclusion bodies. Although a number of general and protein-specific techniques are available, their effectiveness varies widely. We report a novel method for enhancing the solubility of overexpressed proteins. Presence of a dipeptide, glycylglycine, in the range of 100 mM to 1 M in the medium was found to significantly enhance the solubility (up to 170-fold) of the expressed proteins. The method has been validated using mycobacterial proteins, resulting in improved solubilization, which were otherwise difficult to express as soluble proteins in E. coli. This method can also be used to enhance the solubility of other heterologous recombinant proteins expressed in a bacterial system.

Bacterial Proteins↗

PubMed1998

Construction of a mouse hepatitis virus recombinant expressing a foreign gene.

The genome of the coronavirus mouse hepatitis virus (MHV) contains genes which have been shown to be nonessential for viral replication and which could, in principle, be used as sites for the introduction of foreign sequences. We have inserted heterologous genetic material into gene 4 of MHV in order (i) to test the applicability of targeted RNA recombination for site-directed mutagenesis of the MHV genome upstream of the N gene; (ii) to develop further genetic tools for mutagenesis of structural genes other than N; and (iii) to examine the feasibility of using MHV as an expression vector. A DI-like donor RNA vector containing the MHV S gene and all genes distal to S was constructed. Initially, a derivative of this was used to insert a 19-nucleotide tag into the start of ORF 4a of MHV-A59 using the N gene deletion mutant A1b4 as the recipient virus. Subsequently, the entire gene for the green fluorescent protein (GFP) was inserted in place of gene 4. This heterologous gene was shown to be expressed by recombinant viruses but not at levels sufficient to allow detection of fluorescence of viral plaques. Northern blot analysis of transcripts of GFP recombinants showed the expected displacement of the mobility, relative to those of wild-type, of all subgenomic mRNAs larger than mRNA5. An unexpected result of the Northern analysis was the observation that GFP recombinants also produced an RNA species the same size as that of wild-type mRNA4. RT-PCR analysis of the 5' end of this species revealed that it was actually a collection of mRNAs originating from a cluster of 10 different sites, none of which possessed a canonical intergenic sequence. The finding of these aberrant mRNAs, all of nearly the same size as wild-type mRNA4, suggests that long range structure of the MHV genome can sometimes be the sole determinant of the site of initiation of transcription.

Animals↗

PubMed1998

Identification and recombinant expression of a Mycobacterium avium rhamnosyltransferase gene (rtfA) involved in glycopeptidolipid biosynthesis.

The Mycobacterium avium complex is a source of disseminated infections in patients with advanced AIDS. This group of mycobacteria is distinguished by the presence of highly antigenic, surface-exposed glycopeptidolipids, and these glycolipids possess variant oligosaccharide structures that are the chemical basis of the 28 distinct serovars of the M. avium complex. We previously described the ser2 gene cluster, encoding the synthesis of the haptenic oligosaccharide (2, 3-dimethylfucose-rhamnose-6-deoxytalose-) of the serovar 2-specific glycopeptidolipid, and revealed a locus (ser2A) encoding a putative rhamnosyltransferase. Sequencing of the ser2A locus demonstrated the presence of three open reading frames, two of which yielded significant homology to several glycosyltransferases, and the deduced amino acid sequences of these two putative glycosyltransferases had 63% identity. These two genes were expressed in Mycobacterium smegmatis, and the resulting recombinant glycopeptidolipids were characterized by thin-layer chromatography and gas chromatography-mass spectrometry. These analyses demonstrated that only one of these genes, termed rtfA, encoded the rhamnosyltransferase responsible for the transfer of rhamnose to 6-deoxytalose. The identification of rtfA will permit further evaluation of glycopeptidolipid biosynthesis and the construction of isogenic mutants of multiple M. avium complex serovars. Moreover, such mutants will help define the role of glycopeptidolipids in the intracellular survival of these bacteria.

Bacterial Proteins↗

PubMed2004

Recombinant expression systems in the pharmaceutical industry.

In terms of downstream processing efficiency, secretory expression systems offer potential advantages for the production of recombinant proteins, compared with inclusion body forming cytosolic systems. However, for high-volume therapeutics like insulin, the product yields of the majority of the potentially available secretory systems is not yet fully competitive. Current strategies to improve productivity and secretion efficiency comprise: (1) enhancement of gene expression rates, (2) optimization of secretion signal sequences, (3) coexpression of chaperones and foldases, (4) creation of protease deficient mutants to avoid premature product degradation and (5) subsequent breeding and mutagenesis. For the production of non-glycosylated proteins and proteins, which are natively glycosylated but are also pharmacologically active without glycosylation, prokaryotes, which usually lack metabolic pathways for glycosylation, are theoretically the most suitable organisms and offer two alternatives: either Escherichia coli strains are conditioned to be efficient secreters or efficient native secreters like Bacillus species are accordingly developed. To fully exploit the secretory capacity of fungal species, a deeper understanding of their posttranslational modification physiology will be necessary to steer the degree and pattern of glycosylation, which influences both folding and secretion efficiency. Insect and mammalian cells display posttranslational modification patterns very similar or identical to humans, but in view of the entailed expenditures, their employment can only be justified if their modification machinery is required to ensure a desired pharmacological activity.

Animals↗

PubMed2005

Recombinant expression of indolicidin concatamers in Escherichia coli.

Antimicrobial peptides are part of the innate immune system of vertebrates and invertebrates. They are active against gram-negative and gram-positive bacteria, fungi, and protozoa. Currently, most antimicrobial peptides are extracted from host organisms or produced by solid-phase peptide synthesis. Recombinant protein expression in Escherichia coli is a tool for greater production yields at a decreased cost and reduces the use of hazardous materials. We have constructed a concatamer of indolicidin and successfully expressed a fusion product with thioredoxin in E. coli BL21DE3. Codons for methionine residues flanking individual indolicidin genes were incorporated for cyanogen bromide cleavage of the fusion protein and liberation of active monomeric indolicidin. Peptide yields of 150 microg/l monomeric indolicidin were achieved in this first report of recombinant production of indolicidin with demonstrated antimicrobial activity.

Amino Acid Sequence↗

PubMed1994

Enhancement of EIAV replication and disease by immunization with a baculovirus-expressed recombinant envelope surface glycoprotein.

The potential for antibody-dependent enhancement of replication of macrophage/monocyte tropic viruses has posed a significant problem in the development of vaccines for several animal and human viruses and has raised significant concern in the design of potential AIDS vaccines. Using the previously described equine infectious anemia virus/Shetland pony system as a model for HIV-1 vaccine development, we have evaluated the efficacy of a recombinant subunit vaccine containing a baculovirus-expressed envelope surface glycoprotein (gp90) of EIAV. The results of these trials demonstrate not only that the recombinant vaccine failed to protect against infection by standard homologous and heterologous EIAV challenge strains, but that it resulted in a marked enhancement of virus replication and exacerbation of disease in immunized ponies exposed to the heterologous virus strain. Thus, the recombinant EIAV gp90 vaccine provides a novel in vivo model for examining in detail the mechanisms of immune enhancement of a lentivirus infection and for evaluating strategies to avoid the production of deleterious immune responses in AIDS vaccine design.

AIDS Vaccines↗