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Results for “Recombinant expression”

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PubMed1992

Increased frequency of both total and specific monoclonal antibody producing hybridomas using a fusion partner that constitutively expresses recombinant IL-6.

The addition of auxiliary feeder cells or conditioned medium has been shown to augment the yield of mouse hybridomas obtained following the cell-cell fusion of myeloma and B lymphocytes. The addition of one of these factors, interleukin-6 (IL-6) has been found to increase the proportion of hybridomas secreting monoclonal antibodies of desired specificity. As an alternative genetic approach, we have examined the efficacy of a retroviral infectant of Sp2/0 cells that constitutively expresses recombinant murine IL-6 (Sp2/mIL-6) as fusion partner. The results demonstrated that the yields of both viable Ig-secreting hybridomas, and antigen-specific monoclonal antibodies were increased 3-15-fold and 5-9-fold, respectively, with the Sp2/mIL-6 relative to Sp2/0 or Sp2/neo cells as fusion partner. Sp2/mIL-6 cells generated hybridomas with comparable growth rates, stability, and Ig production. The results of staining nascent hybridoma colonies immunohistochemically for Ig production suggest that Sp2/mIL-6 cells as a fusion partner increased the viability and/or stability of nascent hybrid cells that are producing Ig. Thus the Sp2/mIL-6 cells are an improved myeloma parent for the generation of large numbers of antibody-producing hybridomas against specific antigens.

Animals↗

PubMed2000

Immunoadjuvant activities of E. coli- and plasmid-expressed recombinant chicken IFN-alpha/beta, IFN-gamma and IL-1beta in 1-day- and 3-week-old chickens.

In the present study we assessed the capacity of recombinant E. coli- or plasmid-expressed chicken interferons (IFN) and chicken IL-1beta, to exert immunostimulatory activities for humoral immune responses, in day-old and adult chickens. We observed that both recombinant E. coli-expressed chicken IFN-alpha/beta and IFN-gamma facilitated the induction of a primary and also a secondary antibody response, using tetanus toxoid (TT) as a bacterial model antigen, in immunologically mature 3-week-old chickens. In contrast, no improvement of antibody either type of chicken IFN was co-injected with inactivated Infectious Bursal Disease Virus (IBDV) antigen. TT-specific antibody formation was marginally increased by co-injection of recombinant E. coli-expressed chicken IL-1beta. Combined administration of IFN-alpha/beta plus IFN-gamma or IL-beta increased responses to TT in an additive, but not synergistic fashion. Remarkably, no augmentation of antibody responses specific for TT, nor IBDV, was noted in day-old birds, receiving IFN-alpha/beta or IFN-gamma as adjuvant. Also, intramuscular immunization of 3-week-old birds, using plasmids encoding IFN-alpha/beta together with TT protein antigen, significantly increased the speed and magnitude of TT-specific antibody responses. Plasmids encoding chicken IL-beta or IFN-gamma had a minimal or inhibitory effect, respectively. These data indicate a potential for chicken cytokines as immunoadjuvant for particular types of chicken vaccine antigens.

Adjuvants, Immunologic↗

PubMed1993

Protection of goats against peste des petits ruminants with a vaccinia virus double recombinant expressing the F and H genes of rinderpest virus.

Peste des petits ruminants (PPR) is a viral disease of goats and sheep characterized by necrotizing and erosive stomatitis, enteritis and pneumonia. The causative agent, PPRV, is a member of the family Paramyxoviridae and the genus Morbillivirus. Other members of the genus include rinderpest (RPV), measles, canine distemper and phocid distemper viruses. PPR has a very high rate of morbidity and mortality, and effective control of this disease is of economic importance in Africa, Asia and the Middle East. Currently, there is no safe and effective vaccine available against the disease. The tissue culture rinderpest vaccine (TCRV) protects small ruminants against severe disease; there are, however, clinical problems associated with vaccination. This laboratory has recently developed several effective vaccinia virus recombinant vaccines for rinderpest. These vaccines are easy to administer, inexpensive to produce and heat-stable. Goats were vaccinated with a vaccinia virus double recombinant expressing the haemagglutinin and fusion genes of RPV. Although vaccinated animals developed antibodies (neutralizing and ELISA) to RPV, and not to PPRV, they were completely protected against challenge inoculation with virulent PPRV. This would indicate that protection is most probably due to cell-mediated immunity. Use of the rinderpest double recombinant vaccinia virus in areas of the world where PPRV is endemic would aid in the control and eradication of PPR.

Animals↗

PubMed2005

Chondroitinase ABC I from Proteus vulgaris: cloning, recombinant expression and active site identification.

GalAGs (galactosaminoglycans) are one subset of the GAG (glycosaminoglycan) family of chemically heterogeneous polysaccharides that are involved in a wide range of biological processes. These complex biomacromolecules are believed to be responsible for the inhibition of nerve regeneration following injury to the central nervous system. The enzymic degradation of GAG chains in damaged nervous tissue by cABC I (chondroitinase ABC I), a broad-specificity lyase that degrades GalAGs, promotes neural recovery. In the present paper, we report the subcloning of cABC I from Proteus vulgaris, and discuss a simple methodology for the recombinant expression and purification of this enzyme. The originally expressed cABC I clone resulted in an enzyme with negligible activity against a variety of GalAG substrates. Sequencing of the cABC I clone revealed four point mutations at issue with the electron-density data of the cABC I crystal structure. Site-directed mutagenesis produced a clone with restored GalAG-degrading function. We have characterized this enzyme biochemically, including an analysis of its substrate specificity. By coupling structural inspections of cABC I and an evaluation of sequence homology against other GAG-degrading lyases, a set of amino acids was chosen for further study. Mutagenesis studies of these residues resulted in the first experimental evidence of cABC I's active site. This work will facilitate the structure-function characterization of biomedically relevant GalAGs and further the development of therapeutics for nerve regeneration.

Amino Acid Substitution↗

PubMed2002

Rescue of rubella virus replication-defective mutants using vaccinia virus recombinant expressing rubella virus nonstructural proteins.

The genome of rubella virus (RV) is translated into a polyprotein precusor, p200, of the nonstructural proteins (NSPs). This is proteolytically processed by a viral-encoded protease into two mature products, p150 and p90. p150 contains sequence corresponding to the predicted methyltransferase and protease activities, while p90 has sequence for the proposed helicase and RNA-dependent RNA polymerase activities. Processing of p200 is essential for RV viral replication. RV NSPs are responsible for viral RNA replication, in which a full-length negative-strand RNA serves as the intermediate for the replication of positive-strand genomic RNA and the transcription of subgenomic RNA. Previously we demonstrated that p200 synthesizes negative- but not positive-strand RNA, and that cleavage products p150/p90 are required for efficient production of positive-strand RNA. To determine whether p150 or p90 alone or together is involved in positive-strand RNA synthesis, vaccinia virus recombinants expressing individual NSPs were constructed and characterized. These were used in in vivo rescue experiments to complement replication-defective mutants in virus replication. A protease-inactive mutant was rescued by p200 or p150 provided in trans by using vaccinia virus recombinants. Thus this protease can function in trans. Rescue of cleavage-defective mutant by either p200 alone, or p150 plus p90 but not by p150 or p90 alone suggests that p150 and p90 function together as a replication complex in positive-strand RNA synthesis.

Cell Line↗

PubMed2005

[Construction and expression of recombinant plasmid with prostate-specific membrane antigen promoter and enhancer regulating uracil phosphoribosyltransferase gene].

OBJECTIVE: To construct the recombinant expression plasmid pPSMA(EP)-UPRT including UPRT gene that is regulated by PSMA(enhancer/promoter). METHODS: By use of PCR, UPRT gene was amplified from E. coli JM109 genome. Then UPRT gene was cloned into the recombinant expression plasmid pPSMA(enhancer/promoter)-EGFP that is driven by prostate-specific membrane antigen promoter and enhancer. RESULTS: It was found that EGFP was digested by two restriction enzymes from the recombinant expression plasmid pPSMA(enhancer/promoter)-EGFP, and UPRT gene was linked with the recombinant expression plasmid by T4 DNA ligase. We succeeded in constructing the recombinant expression plasmid pPSMA(enhancer/promoter)-UPRT. The recombinant plasmid sequences were verified, and the expression in vitro was measured by MTT. CONCLUSION: The recombinant expression plasmid pPSMA(enhancer/promoter)-UPRT is regulated targetly by prostate-specific membrane antigen promoter and enhancer. It is, of importance to us in studying the UPRT/5-FU for gene therapy of prostate cancer, especially suicide gene therapy (CD/5-FC system).

Adenocarcinoma↗

PubMed2006

Yersinia enterocolitica type III secretion chaperone SycD: recombinant expression, purification and characterization of a homodimer.

Yersinia species pathogenic to human benefit from a protein transport machinery, a type three secretion system (T3SS), which enables the bacteria to inject effector proteins into host cells. Several of the transport substrates of the Yersinia T3SS, called Yops (Yersinia outer proteins), are assisted by specific chaperones (Syc for specific Yop chaperone) prior to transport. Yersinia enterocolitica SycD (LcrH in Yersinia pestis and Yersinia pseudotuberculosis) is a chaperone dedicated to the assistance of the translocator proteins YopB and YopD, which are assumed to form a pore in the host cell membrane. In an attempt to make SycD amenable to structural investigations we recombinantly expressed SycD with a hexahistidine tag in Escherichia coli. Combining immobilized nickel affinity chromatography and gel filtration we obtained purified SycD with an exceptional yield of 120mg per liter of culture and homogeneity above 95%. Analytical gel filtration and cross-linking experiments revealed the formation of homodimers in solution. Secondary structure analysis based on circular dichroism suggests that SycD is mainly composed of alpha-helical elements. To prove functionality of purified SycD previously suggested interactions of SycD with Yop secretion protein M2 (YscM2), and low calcium response protein V (LcrV), respectively, were reinvestigated.

Antigens, Bacterial↗

PubMed1999

Cloning and recombinant expression of a novel mouse-secreted phospholipase A2.

Secreted phospholipases A2 (sPLA2s) form a class of structurally related enzymes that are involved in a variety of physiological and pathological effects including inflammation and associated diseases, cell proliferation, cell adhesion, and cancer, and are now known to bind to specific membrane receptors. Here, we report the cloning and expression of a novel sPLA2 isolated from mouse thymus. Based on its structural features, this sPLA2 is most similar to the previously cloned mouse group IIA sPLA2 (mGIIA sPLA2). As for mGIIA sPLA2, the novel sPLA2 is made up of 125 amino acids with 14 cysteines, is basic (pI = 8.71) and its gene has been mapped to mouse chromosome 4. However, the novel sPLA2 has only 48% identity with mGIIA and displays similar levels of identity with the other mouse group IIC and V sPLA2s, indicating that the novel sPLA2 is not an isoform of mGIIA sPLA2. This novel sPLA2 has thus been called mouse group IID (mGIID) sPLA2. In further contrast with mGIIA, which is found mainly in intestine, transcripts coding for mGIID sPLA2 are found in several tissues including pancreas, spleen, thymus, skin, lung, and ovary, suggesting distinct functions for the two enzymes. Recombinant expression of mGIID sPLA2 in Escherichia coli indicates that the cloned sPLA2 is an active enzyme that has much lower specific activity than mGIIA and displays a distinct specificity for binding to various phospholipid vesicles. Finally, recombinant mGIID sPLA2 did not bind to the mouse M-type sPLA2 receptor, while mGIIA was previously found to bind to this receptor with high affinity.

Amino Acid Sequence↗

PubMed2002

Elastin as a self-organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self-assembly of elastin.

Elastin is the major extracellular matrix protein of large arteries such as the aorta, imparting characteristics of extensibility and elastic recoil. Once laid down in tissues, polymeric elastin is not subject to turnover, but is able to sustain its mechanical resilience through thousands of millions of cycles of extension and recoil. Elastin consists of ca. 36 domains with alternating hydrophobic and cross-linking characteristics. It has been suggested that these hydrophobic domains, predominantly containing glycine, proline, leucine and valine, often occurring in tandemly repeated sequences, are responsible for the ability of elastin to align monomeric chains for covalent cross-linking. We have shown that small, recombinantly expressed polypeptides based on sequences of human elastin contain sufficient information to self-organize into fibrillar structures and promote the formation of lysine-derived cross-links. These cross-linked polypeptides can also be fabricated into membrane structures that have solubility and mechanical properties reminiscent of native insoluble elastin. Understanding the basis of the self-organizational ability of elastin-based polypeptides may provide important clues for the general design of self-assembling biomaterials.

Amino Acid Sequence↗

PubMed1999

The use of elements of the E. coli Ntr-system for the design of an optimized recombinant expression system for high cell density cultivations.

The inducible glnA promoter 2 of the E. coli glutamine synthetase gene is suitable as an expression unit for the production of recombinant proteins at low and high cell densities. It is active when the concentration of ammonium as the sole nitrogen source in the culture medium is below 1 mM. This nitrogen regulatory system was optimized by introduction of expression cassettes consisting of additional elements of the ntr-system. These artificial constructions result in enhanced recombinant gene expression in the production phase. Furthermore, the basic recombinant protein level during the growth phase is reduced due to a tighter promoter control. A three- to four-fold higher accumulation of chloramphenicol-acetyltransferase (as reporter protein) and of anti-EGF-receptor miniantibodies was achieved by increasing the amount of the final regulator molecule NtrC approximately P via plasmidal co-expression of the ntrC gene. The introduction of a modified glnA promoter 1 inverse to glnAp2 lowered the basic activity of glnAp2 to about one half. It is assumed that under nitrogen excess conditions sigma 70-RNA polymerase binds at glnAp1 and thereby prevents most of the binding of sigma 54-RNA polymerase at glnAp2. The optimized expression systems were successfully applied in low and high cell density cultivations. In the fed-batch phase of high cell density cultivations recombinant protein formation was induced through external nitrogen limitation under FIA-controlled concentration of glucose as carbon source.

Antibodies↗

PubMed1992

Purification of liver aldehyde dehydrogenase by p-hydroxyacetophenone-sepharose affinity matrix and the coelution of chloramphenicol acetyl transferase from the same matrix with recombinantly expressed aldehyde dehydrogenase.

p-Hydroxyacetophenone was coupled to epoxy-activated Sepharose 6B to generate an affinity chromatographic matrix to purify aldehyde dehydrogenase. Purified beef liver mitochondrial aldehyde dehydrogenase specifically bound to the support and could be eluted with p-hydroxyacetophenone. A post-ammonium sulfate (30-55%) fraction of bovine liver was applied to the affinity gel column and aldehyde dehydrogenase was effectively purified, although not to complete homogeneity, indicating the potential selectivity of the matrix. Both beef liver cytosolic and mitochondrial aldehyde dehydrogenase bound to the column. A post-Cibacron blue Sepharose Cl-6B affinity-fractionated liver mitochondrial aldehyde dehydrogenase was purified to complete homogeneity by p-hydroxyacetophenone-Sepharose, thus eliminating the need for the isoelectric focusing step often employed. p-Hydroxyacetophenone was found to be a competitive inhibitor against propionaldehyde and noncompetitive against NAD. Escherichia coli lysates of recombinantly expressed aldehyde dehydrogenase were purified from E. coli lysates with one major 25-kDa protein contaminant also binding to the column, as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The 25-kDa contaminant was found to be chloramphenicol acetyl transferase from sequence analysis and binding studies.

Acetophenones↗

PubMed1989

Development of an enzyme immunoassay using recombinant expressed antigen to detect hepatitis delta virus antibodies.

Two generic enzyme immunoassays (EIAs) were developed for detection of anti-hepatitis delta virus antibodies (anti-HD) and compared with a commercially available radioimmunoassay. Both generic assays were configured as blocking assays and used hepatitis delta antigen (HDAg) derived from infected chimpanzee liver (EIA-1) or from Escherichia coli transformed with a plasmid containing an insert from within an open reading frame encoding HDAg (EIA-2). Absolute sensitivity was ascertained by endpoint titration, which demonstrated essentially identical endpoints for EIA-1 and EIA-2. The absolute sensitivities of the EIAs were approximately four times greater than that of the radioimmunoassay. Specificity and sensitivity were ascertained by testing a panel of 176 serum specimens by each assay. The specimens were selected to represent a panel composed of sera from individuals with or without markers of viral hepatitis as follows: (i) serologically confirmed by exclusion as posttransfusion non-A, non-B hepatitis; (ii) acute or chronic hepatitis B virus infection, positive for hepatitis B surface antigen; (iii) resolved hepatitis B virus infection, positive for anti-hepatitis B surface antigen; (iv) acute hepatitis A virus infection, positive for anti-hepatitis A virus immunoglobulin M; and (v) normal human sera. All three assays for anti-HD gave similar specificity and sensitivity values. In conclusion, the recombinant expressed HDAg can replace antigen derived from infected liver tissue as a diagnostic reagent used to configure an EIA for detection of anti-HD. Furthermore, the results suggest that the expressed antigen contains the important immunodominant epitope(s).

Antibodies, Viral↗

PubMed1984

Construction of live vaccines using genetically engineered poxviruses: biological activity of vaccinia virus recombinants expressing the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D.

Potential live vaccines using recombinant vaccinia viruses have been constructed for both hepatitis B and herpes simplex. These recombinant vaccinia viruses express cloned genes of the hepatitis B virus surface antigen (HBsAg) or the glycoprotein D from herpes simplex virus (HSV-gD). The HBsAg synthesized in vitro under the regulation of vaccinia virus is secreted from infected cells as a particle of approximately equal to 22 nm diameter with a density of 1.2 g/ml as determined on CsCl gradients. Inoculation of rabbits with the recombinant vaccinia virus that expresses the HBsAg elicits the production of high-titered antibodies. Synthesis of the HSV-gD was detected in tissue culture by radioimmunoassay on unfixed cells, suggesting that the HSV-gD synthesized by the recombinant vaccinia virus is membrane associated. Inoculation of rabbits with the recombinant vaccinia virus expressing HSV-gD resulted in the production of antibodies that reacted with authentic HSV-gD as detected by radioimmunoassay. Furthermore, the anti-serum was shown by plaque-reduction assay to neutralize the infectivity of herpes simplex virus. Immunization of mice with the vaccinia recombinant expressing HSV-gD gave complete protection on subsequent challenge with lethal doses of live herpes simplex virus.

Animals↗

PubMed2004

Establishment and application of in vitro membrane potential assays in cell lines with endogenous or recombinant expression of ATP-sensitive potassium channels (Kir6.2/SUR1) using a fluorescent probe kit.

The flow of current through the adenosine triphosphate (ATP)-sensitive potassium channel (K(ATP)) of the isoform Kir6.2/SUR1 regulates the resting membrane potential in the pancreatic beta-cell. In combination with the cellular glucose metabolism, it is an important minute-to-minute regulator of insulin secretion and whole-body glucose homeostasis. The same K(ATP) isoform is further reported to be present in glucagon-secreting alpha-cells, intestinal L-cells, and glucose-responsive neurons in the hypothalamus. All in all, this makes Kir6.2/SUR1 an interesting drug target. Using a commercially available fluorescent membrane potential probe kit and a conventional 96-well fluorescence plate reader, the authors have developed and established qualitative membrane potential assays used to screen for potassium channel closers (KCCs) and openers (KCOs) in insulin- and glucagon-secreting cell lines as well as in cells with recombinant expression of the human Kir6.2/SUR1 channel complex. Both glucose- and KCC-induced depolarization could be demonstrated. The magnitudes of these responses and KCO-induced repolarization at high glucose displayed some variation between the different cell lines but a similar rank order of test compounds. Some cell types required the presence of a KCC, such as tolbutamide, to display significant effects of KCOs. The authors find that robust and reliable functional in vitro assays compatible with medium-throughput screening and high-throughput screening can be developed as a base for finding new, more potent, and isoform-selective KCCs and KCOs.

Cell Line↗

PubMed2026

Molecular Cloning, Recombinant Expression, and In Silico Structural Analysis of Cu/Zn-Superoxide Dismutase from Trachyspermum ammi.

Superoxide dismutase (SOD) is an essential antioxidant metalloenzyme that is critical for the cellular defense against oxidative damage, as it scavenges superoxide radicals and maintains the redox status. Cytosolic Cu/Zn-SOD is particularly important in the regulation of oxidative stress among different isoforms in higher plants. While Cu/Zn-SODs from several plant species have been characterized, molecular information is limited for Trachyspermum ammi, a medicinally important member of a family Apiaceae with antioxidant potential.In the present study, an integrated molecular and in silico approach has been taken to clone and analyze a Cu/Zn type SOD gene from T. ammi to get insight into its structural and evolutionary characteristics. PCR amplification yielded an open reading frame of 456 bp encoding a protein of 152 amino acids. Sequence analysis showed that plant Cu/Zn-SODs, especially those from Daucus carota, were highly similar to one another (about 90-95%).Multiple sequence alignment confirmed the presence of conserved catalytic motifs and metal-binding histidine residues, both of which are crucial for enzymatic function. Physicochemical analysis predicted the protein to be stable, hydrophilic and compatible with cytosolic localization. The analysis of secondary structure indicated a predominance of β-strands, consistent with the conserved β-barrel architecture of plant Cu/Zn-SODs.The three-dimensional structure was built by homology modeling using a closely related plant Cu/Zn-SOD template with high sequence identity. Structural validation demonstrated an acceptable stereochemical quality with 86.3% residues in the favored region of Ramachandran plot, satisfactory ERRAT and Verify3D scores, and a low RMSD value of 0.104 Å on structural superimposition. Phylogenetic analysis placed the enzyme in the Apiaceae lineage, suggesting evolutionary conservation among related plant species. In conclusion, this study presents the first molecular and structural characterization of Cu/Zn-SOD from T. ammi and confirms the existence of a conserved structural framework typical of plant Cu/Zn-SODs. These results provide a basis for further studies concerning recombinant expression, enzymatic validation and potential relevance in antioxidant and plant stress biology.

Cloning, Molecular↗

PubMed2003

Establishment of a recombinant expression system for connective tissue growth factor (CTGF) that models CTGF processing in utero.

Connective tissue growth factor (CTGF) stimulates cell proliferation, migration, adhesion and extracellular matrix production, and functions in processes such as development, differentiation, angiogenesis, implantation, wound healing and fibrosis. CTGF is a 38 kDa protein that comprises four discrete structural modules (modules 1-4) but is susceptible to limited proteolysis in utero yielding bioactive isoforms that comprise either modules 3 and 4 (16-20 kDa) or module 4 (10 kDa). Here we report the development of a stable cell line, termed DB1, that was generated by transfecting cDNA encoding full-length human CTGF into Chinese hamster ovary cells that were mutant for heparin sulphate and chondroitin sulphate. DB1 cells produced 38 kDa CTGF and low molecular mass CTGFs that had N-termini between modules 2 and 3 at Ala(181) (20 kDa), Leu(184) (18 kDa) or Ala(197) (16 kDa) or between modules 3 and 4 at Gly(253) (10 kDa). CTGF was exported from DB1 cells as early as 5 min after synthesis and all isoforms were readily purified from conditioned medium by sequential steps of heparin affinity, cation exchange, and reverse-phase chromatography. The 38 kDa CTGF was faithfully glycosylated and underwent limited proteolysis in the presence of thrombin, kallikrein or uterine fluids, the last of which was antagonized by anti-thrombin III. All CTGF isoforms promoted cell adhesion, mitosis and epithelial transdifferentiation in vitro as well as subcutaneous fibrosis in vivo. The establishment of this recombinant expression system allows for mass-scale production of all previously reported uterine CTGF isoforms, demonstrates that module 4 contains functional domains involved in a broad range of biological activities, and will facilitate studies of CTGF processing in vitro.

Animals↗

PubMed2006

One-step purification of bacterially expressed recombinant transducin alpha-subunit and isotopically labeled PDE6 gamma-subunit for NMR analysis.

Interactions between the transducin alpha-subunit (Galpha(t)) and the cGMP phosphodiesterase gamma-subunit (PDEgamma) are critical not only for turn-on but also turn-off of vertebrate visual signal transduction. Elucidation of the signaling mechanisms dominated by these interactions has been restrained by the lack of atomic structures for full-length Galpha(t)/PDEgamma complexes, in particular, the signaling-state complex represented by Galpha(t).GTPgammaS/PDEgamma. As a preliminary step in our effort for NMR structural analysis of Galpha(t)/PDEgamma interactions, we have developed efficient protocols for the large-scale production of recombinant Galpha(t) (rGalpha(t)) and homogeneous and functional isotopically labeled PDEgamma from Escherichia coli cells. One-step purification of rGalpha(t) was achieved through cobalt affinity chromatography in the presence of glycerol, which effectively removed the molecular chaperone DnaK that otherwise persistently co-purified with rGalpha(t). The purified rGalpha(t) was found to be functional in GTPgammaS/GDP exchange upon activation of rhodopsin and was used to form a signaling-state complex with labeled PDEgamma, rGalpha(t). GTPgammaS/[U-13C,15N]PDEgamma. The labeled PDEgamma sample yielded a well-resolved 1H-15N HSQC spectrum. The methods described here for large-scale production of homogeneous and functional rGalpha(t) and isotope-labeled PDEgamma should support further NMR structural analysis of the rGalpha(t)/PDEgamma complexes. In addition, our protocol for removing the co-purifying DnaK contaminant may be of general utility in purifying E. coli-expressed recombinant proteins.

Amino Acid Sequence↗

PubMed2003

Development of a cocktail of recombinant-expressed human rabies virus-neutralizing monoclonal antibodies for postexposure prophylaxis of rabies.

To provide a cost-effective and safe replacement for human rabies immunoglobulin (HRIG), we used DNA recombinant technology to express 3 human rabies virus-neutralizing human monoclonal antibodies (huMAbs) in a rhabdovirus vector (RhV). Infection of either baby hamster kidney cells or CHO cells, with the resulting RhV-huMAb recombinant viruses, yielded high-level production (< or =40 micro g/mL/48 h) of RhV recombinant-expressed huMAbs (rhuMAbs) that differ in both isotype and epitope-recognition specificity. A cocktail of these rhuMAbs neutralizes several fixed and street wild-type rabies viruses (RVs). Mice and hamsters treated only once with this rhuMAb cocktail after infection with a lethal dose of RV were protected. In the mouse models, the postexposure prophylaxis (PEP) efficacy obtained with the rhuMAb cocktail was comparable to that obtained with HRIG, a finding strongly suggesting that rhuMAbs should be given serious consideration for use in future PEP of humans.

Animals↗