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At least 19 recordsLinked to original sources
PubMed1993

Detection of human T-lymphotropic virus type-I/II env antibodies by immunoassays using recombinant fusion proteins.

Two recombinant fusion proteins representing the C-terminus of the envelope glycoprotein of HTLV-I (rEnv-93(201-440)) and the C-terminus of the external glycoprotein (RE-3(165-306)) were tested in a Western blot (WB) assay for their ability to detect the presence of env antibodies in serum specimens from HTLV-I (n = 27) and HTLV-II (n = 22) infected individuals. The rEnv-93 reacted with 27 (100%) of 27 HTLV-I-infected specimens and 19 (86%) of 22 of HTLV-II-infected specimens. In contrast, RE-3 reacted with 25 (93%) of 27 HTLV-I-infected specimens, and only six (27%) of 22 HTLV-II-infected specimens, thus demonstrating predominant reactivity with HTLV-I compared with HTLV-II. Because of the high sensitivity of rEnv-93 reactivity in both HTLV-I and HTLV-II, the specificity of this env protein was evaluated in specimens with isolated gag reactivity (HTLVind). Of the 44 HTLVind specimens, four (9%) demonstrated reactivity to rEnv-93 in WB assay. We, therefore, conclude that although rEnv-93 is highly sensitive for detection of env protein, it has the potential to yield some false-positive reactions presumably due to the conserved nature of retroviral transmembrane epitopes.

Amino Acid Sequence↗
PubMed1995

Antibodies prevalence against HPV-6b and -16 recombinant fusion proteins in Korean patients with cervical neoplasia.

OBJECTIVES: This study was to investigate the adjunctive diagnostic utility of HPV fusion proteins from the sera in the patients with cervical neoplasia. Immunologic researches on host factors in HPV infection could contribute to better understanding of patho-physiologic mechanisms of cervical carcinogenesis, and provide valuable information for HPV vaccine development. METHODS: Prevalence of antibodies against human papillomavirus (HPV) in sera of the patients with carcinoma (n = 81), precancers (n = 25) of uterine cervix and normal controls (n = 40) in Korean women were investigated by Western blot immunoassay using partially purified TrpE fusion recombinant proteins for HPV-6b and HPV-16. RESULTS: In 81 patients with cervical cancer, 15 (19%) and 54 (67%) patients were positive for antibodies to at least one of the tested recombinant proteins related to HPV-6b or HPV-16, respectively. And in 25 patients with HPV-related squamous intraepithelial lesion (SIL) of cervix, 10 were positive for at least one of the tested antibodies for HPV-6b gene products (40%) and 11 were positive for HPV-16 related antibodies (44%). The prevalence rates of antibodies for HPV-6b in the sera of SIL patients were significantly higher than those from the sera of cervical cancer patients (p < 0.05). There was an inverse correlation between increasing the positivity against HPV-16 E6 protein and progression of cervical lesions, but there was no significant correlation between the positivities of the other antibodies to HPV-16 and the severity of disease. CONCLUSION: These results suggest that the recombinant proteins related to HPV-6b and HPV-16 could be useful in evaluation of the patients with HPV-related cervical lesions and these might play an adjunctive role in diagnosis and management of cervical neoplasia.

Antibodies, Viral↗
PubMed1998

Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system.

We used the recombinant baculovirus/insect cell system to express two soluble forms of the mouse Fas receptor (mFasR) extracellular domain (ECD): a monomer comprising the entire ligand-binding portion of mFasR followed by a carboxy-terminal hexa-histidine extension aiding purification by immobilized metal affinity chromatography and an immunoadhesin in which the same 148 residues were fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain. Both constructs harboured a 24 base pairs insertion placed upstream of the initiating ATG [Peakman, Charles, Sydenham, Gewert, Page, and Makoff (1992) Nucleic Acids Res. 20, 6111-6112]. Despite its hexa-histidine extension, the monovalent recombinant protein from crude culture media failed to bind immobilized Ni2+ unless proteins were first precipitated twice by ammonium sulphate. The overall procedure then yielded approximately 10mg/l of protein which could be purified to near homogeneity using two additional chromatographic steps. The glycosylated polypeptide migrated as a band of Mr=(21-31) x 10(3) in SDS/PAGE and was monomeric in physiological buffers. Under non-reducing conditions, denaturation in 6 M guanidinium chloride was reversible after slow removal of the denaturing agent. The mFasR immunoadhesin was secreted (approximately 5-10 mg/l) as a disulphide-linked homodimer, and endowed with ligand-binding activity since it could bind FasL on the surface of D11S, FasL-expressing cells. When tested for their ability to inhibit FasR-dependent cell lysis, the soluble dimeric immunoadhesin markedly inhibited FasL-mediated cytotoxicity (IC50 approximately 30 nM), and was approximately 6 times as effective as its monomeric counterpart.

Animals↗
PubMed2003

Precise and efficient cleavage of recombinant fusion proteins using mammalian aspartic proteases.

Expression of recombinant proteins as translational fusions is commonly employed to enhance stability, increase solubility and facilitate purification of the desired protein. In general, such fusion proteins must be cleaved to release the mature protein in its native form. The usefulness of the procedure depends on the efficiency and precision of cleavage and its cost per unit activity. We report here the development of a general procedure for precise and highly efficient cleavage of recombinant fusion proteins using the protease chymosin. DNA encoding a modified pro-peptide from bovine chymosin was fused upstream of hirudin, carp growth hormone, thioredoxin and cystatin coding sequences and expressed in a bacterial Escherichia coli host. Each of the resulting fusion proteins was efficiently cleaved at the junction between the pro-peptide and the desired protein by the addition of chymosin, as determined by activity, N-terminal sequencing and mass spectrometry of the recovered protein. The system was tested further by cleavage of two fusion proteins, cystatin and thioredoxin, sequestered on oilbody particles obtained from transgenic Arabidopsis seeds. Even when the fusion protein was sequestered and immobilized on oilbodies, precise and efficient cleavage was obtained. The precision, efficiency and low cost of this procedure suggest that it could be used in larger scale manufacturing of recombinant proteins which benefit from expression as fusions in their host organism.

Amino Acid Sequence↗
PubMed2003

The role of the Aspergillus niger furin-type protease gene in processing of fungal proproteins and fusion proteins. Evidence for alternative processing of recombinant (fusion-) proteins.

We have characterized growth and protein processing characteristics of Aspergillus niger strains carrying a disrupted allele of the previously cloned and characterized kexB gene [Appl. Environ. Microbiol. 66 (2000) 363] encoding a furin-type endoprotease. Deletion of the single-copy gene confirms it to be non-essential but disruptant strains exhibit a morphologically distinct phenotype characterized by hyperbranching. Processing of homologous pro-proteins and fusion proteins comprised of a heterologous protein fused down-stream of glucoamylase and separated at the fusion junction by an endoproteolytic cleavage site was compared in wildtype and mutant strains of A. niger. We show that maturation of the native glucoamylase requires KexB, whereas maturation of aspergillopepsin does not. The processing of fusion proteins carrying Lys-Arg requires KexB, although alternative endoproteases are capable of cleaving protein fusions at sites adjacent to Lys-Arg.

Aspergillus niger↗
PubMed2002

Immobilization and utilization of the recombinant fusion proteins trypsin-streptavidin and streptavidin-transglutaminase for modification of whey protein isolate functionality.

A method was developed for the production of a hydrolyzed/polymerized whey protein derivative with altered solution and gelation properties using a combination of recombinant DNA and immobilized enzyme technologies. The recombinant fusion proteins trypsin-streptavidin (TrypSA) and streptavidin-transglutaminase (cSAcTG) were produced in Escherichia coli, extracted, and then immobilized by selective adsorption on biotinylated controlled-pore glass. Recirculation through a TrypSA reactor induced limited proteolysis of whey proteins. Hydrolysates were then recirculated through a cSAcTG reactor for incremental periods of time to arrive at increasing degrees of polymerization. The polymers were subsequently analyzed for viscosity/flow behavior, gelation properties, and fracture properties using shear rate ramps/intrinsic viscosity, small-strain oscillatory rheology, and vane viscometry, respectively. By combining limited proteolysis with controlled cross-linking, it was possible to create derivatives of whey proteins with enhanced functional properties. Increases in the degree of whey protein modification were correlated with greater apparent viscosity and intrinsic viscosity, lowered gel point temperatures, and stronger, more brittle gels. This method allowed for recycling of the enzyme, eliminated the requirement for a downstream inactivation step, and permitted control over the extent of modification. Utilization of a similar process may allow for the production of designer proteins engineered with specific functionalities.

Adsorption↗
PubMed1996

Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases.

Obtaining high quality protein crystals remains a rate-limiting step in the determination of three-dimensional X-ray structures. A frequently encountered problem in this respect is the high or heterogeneous carbohydrate content of many eukaryotic proteins. A number of reports have demonstrated the use of enzymatic deglycosylation in the crystallization of certain glycoproteins. Although this is an attractive tool, there are some problems that hinder the more widespread use of glycosidases in crystallization. First, commercially available glycosidases are relatively expensive, which virtually prohibits their use on a large scale. Second, the glycosidase must be removed from the glycoprotein of interest following deglycosylation, which is not always straightforward. To circumvent these problems we have cloned the two most generally useful glycosidases, peptide-N-glycosidase F and endoglycosidase F1 from Flavobacterium meningosepticum, as fusion proteins with glutathione S-transferase. The fusion not only allows rapid purification of these enzymes from Escherichia coli cell extracts, but also permits rapid removal from target proteins following deglycosylation. We have used these enzymes to obtain crystals of phytase from Aspergillus ficuum and acid phosphatase from Aspergillus niger and to obtain a new crystal form of recombinant human renin.

Glycoside Hydrolases↗
PubMed1997

Introduction of spacer peptides N-terminal to a cleavage recognition motif in recombinant fusion proteins can improve site-specific cleavage.

To improve site-specific cleavage of a methionyl porcine growth hormone [[Met1]-pGH(1-46)-IGF-II] fusion protein by the enzyme H64A subtilisin, a series of flexible, unstructured spacer peptides were introduced N-terminal to the cleavage site. When enzymatic digestion preceded refolding of the fusion proteins, IGF-II could only be liberated from substrates which contained spacer peptides. Compared with the parent construct, the yield of IGF-II from refolded fusion proteins containing spacers was improved up to two-fold. Furthermore, this cleavage rate was improved by removing a competing protease recognition motif from the fusion partner. These data show that fusion partners can influence site-specific proteolysis of fusion proteins. Introduction of flexible spacers between the moieties can alleviate these interactions.

Amino Acid Sequence↗
PubMed1993

Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins.

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1 protein into a recombinant vaccine.

Amino Acid Sequence↗
PubMed1993

Redistribution of a murine humoral immune response following removal of an immunodominant B cell epitope from a recombinant fusion protein.

Immunization of different mice strains with a recombinant fusion protein composed of the vector-encoded N-terminal leader peptide CroLac (containing lambda Cro and LacI fragments) and a part of the transmembrane protein of HIV-1 (gp41) led to a high anti-CroLac humoral immune response. A detailed analysis of this response revealed the presence of an immunodominant, linear B cell epitope localized near the C-terminus of the CroLac fragment. The immune response seemed to be biased towards this epitope since few or no monoclonal antibodies (mAb) could be generated against the remaining part of CroLac and the gp41 fragment. Upon removal of the immunodominant region from the fusion protein the immune response was redirected and spread over the previously non-immunogenic regions. Consequently, we report a model system in which an immunodominant B cell epitope biases the immune response away from less immunogenic epitopes on the same molecule.

Amino Acid Sequence↗
PubMed2005

Incorporating protein transduction domains (PTD) within intracellular proteins associated with the 'stemness' phenotype. Novel use of such recombinant 'fusion' proteins to overcome current limitations of applying autologous adult stem cells in regenerative medicine?

Adult stem cells originating from post-natal tissues hold tremendous promise in regenerative medicine. Nevertheless, there are several deficiencies of adult stem cells that would limit their application in transplantation therapy, in particular their relative scarcity, restricted multi-potency and limited proliferative capacity in vitro. A possible approach to overcome these limitations would be to genetically modulate adult stem cells to strongly express genes that are closely associated with the 'stemness' phenotype. Overwhelming safety concerns would preclude the direct application of recombinant DNA technology in genetic modulation. Moreover, constitutive expression of 'stemness' genes would prevent adult stem cells from participating in tissue/organ regeneration upon transplantation. A novel alternative would be to incorporate protein transduction domains within intracellular proteins (i.e. transcription factors) that are associated with the 'stemness' phenotype. Such recombinant fusion proteins would then have the ability to translocate across the cell membrane and be internalized within the cytosol, thereby enabling them to exert a gene-modulatory effect on the cell, without any permanent genetic alteration. This would be particularly useful for maintaining the 'stemness' of adult stem cell populations during extensive ex vivo proliferation, to generate adequate cell numbers for transplantation therapy.

Adult↗
PubMed1987

Activity of a recombinant fusion protein between transforming growth factor type alpha and Pseudomonas toxin.

The transforming growth factor type alpha gene has been fused to a modified Pseudomonas toxin gene from which the cell-recognition domain has been deleted. The chimeric gene has been expressed in Escherichia coli, and the chimeric protein, PE40-TGF-alpha, has been highly purified. PE40-TGF-alpha kills cells expressing epidermal growth factor receptors and has little activity against cells with few receptors. This chimeric protein might be useful in treating cancers that contain high numbers of epidermal growth factor receptors.

ADP Ribose Transferases↗
PubMedDate not supplied

Inhibition of established tumor growth in syngeneic mice by local inoculation of engineered mouse myeloma cells secreting a recombinant fusion protein RM4/TNF.

Mouse myeloma cell line VKCK/RM4-TNF secreting the recombinant fusion protein RM4/TNF was used to study the relationship between tumor necrosis factor (TNF) secretion of tumor cells and its tumorigenicity, and to study the potential mechanisms responsible for the antitumor immune response. To evaluate tumorigenicity, 5 x 10(5) viable TNF-secreting VKCK/RM4-TNF and non-TNF-secreting VKCK tumor cells were subcutaneously injected into BALB/c mice. Tumor progression or regression was evaluated 2 weeks after tumor inoculation. Our animal studies showed that RM4/TNF secretion by VKCK/RM4-TNF tumor cells curtailed its tumorigenicity in BALB/c mice and induced a long-term, protective immune response against a subsequent challenge of the parental VKCK tumor cells. Our animal studies in T-cell subset-depleted BALB/c mice and in T-cell-deficient nude mice demonstrated that both CD4+ and CD8+ T cells play a major role in the reduction of tumorigenicity. In addition, our results further showed that local inoculation of irradiated VKCK/RM4-TNF cells secreting TNF was able to significantly inhibit the established VKCK tumors in syngeneic mice. This study thus highlights the potential utility of engineered tumor cells secreting RM4/TNF in cancer gene therapy.

Animals↗
PubMed1996

Influence of adjuvants on protection induced by a recombinant fusion protein against malarial infection.

Previously, we described a protective immune response induced by the carboxyl-terminal region of the merozoite surface protein-1 (MSP-1) from the rodent malarial parasite Plasmodium yoelii yoelii 17XL, expressed as a fusion protein and designated glutathione S-transferase (GST)-PYC2. We also demonstrated that the humoral response induced by GST-PYC2 was the primary mechanism by which immunized animals controlled their blood-stage infections. We have now examined the influence of several adjuvants on the immune response to the GST-PYC2 fusion protein. While alum, Freund's adjuvant, Ribi adjuvant system, and TiterMax were efficacious in eliciting a protective response with GST-PYC2 in BALB/c mice, saponin failed to induce protection, although significant levels of PYC2-specific antibodies were produced in all immunized animals. This protection depended on the mouse strain since immunization of Swiss Webster mice with GST-PYC2 in alum did not produce levels of PYC2-specific antibodies comparable to those in BALB/c mice nor did it induce any demonstrable level of protection against parasite challenge. Swiss Webster mice were protected, however, when immunized with GST-PYC2 in other adjuvants. Immunization with PYC2, isolated free of GST induced lower levels of antigen-specific antibody; only those animals given PYC2 in Freund's adjuvant demonstrated a significant degree of protection, suggesting the possibility (of additional cellular effector mechanisms. These findings demonstrate that adjuvant, host genotype, and the fine specificity of the response significantly influence the protection induced by the carboxyl terminus of MSP-1 in vivo and illustrate the need to consider these factors in evaluating MSP-1 as a vaccine component.

Adjuvants, Immunologic↗
PubMed1996

The omega-hydroxlyation of lauric acid: oxidation of 12-hydroxlauric acid to dodecanedioic acid by a purified recombinant fusion protein containing P450 4A1 and NADPH-P450 reductase.

The recombinant fusion protein rF450[mRat4Al/mRatOR]L1, containing the heme domain of P450 4A1 and the flavin domains of NADPH-P450 reductase, when incubated with dilaurylphosphatidylcholine (DLPC), Chaps, cytochrome b5, and a 20-fold excess of purified NADPH-P450 reductase, catalyzes the omega- oxidation of lauric acid at a rate of about 300 nmol/min/nmol P450. This is the first report of a mammalian P450 enzyme with such a high turnover number. The resultant 12-hydroxydodecanoic acid [12-hydroxylauric acid (12-OH LA)] is further oxidized by the P450 oxygenase reaction to dodecanedioic acid (decane-1,10-dicarboxylic acid) via 12,12-dihydroxydodecanoic acid. Spectral binding studies show that 12-OH LA inhibits the binding of lauric acid to the active site of P450 with a Ki of about 1.9 microM. The construction and expression of recombinant P450 4A1 containing a six-member polyhistidine domain at the carboxy-terminus of the protein is described. Reconstitution experiments with this purified recombinant P450 4A1, DLPC, Chaps, b5, and purified NADPH-P450 reductase show results similar to those obtained with the purified fusion protein, albeit at lower turnover rates. The requirement for normal-phase HPLC in resolving the metabolites formed during lauric acid metabolism is demonstrated.

Animals↗
PubMed1992

An ELISA sandwich capture assay for recombinant fusion proteins containing glutathione-S-transferase.

A sandwich capture ELISA technique is presented for detection of recombinant proteins sharing a common affinity domain or reagents such as antibodies that bind to these proteins. An activated carrier protein (BSA) is modified with reduced glutathione (GT), forming an affinity capture reagent for glutathione-S-transferase (GST) and recombinant fusion proteins bearing the GST moiety. GT-BSA is immobilized on microtiter plates, and a sandwich is formed consisting of the recombinant fusion protein, reactive antibodies, and detection antibodies. An example is given that demonstrates that this format yields equivalent results to a conventional ELISA test with a panel of newly diagnosed diabetic sera reacting with an islet cell autoantigen.

Chromatography, Affinity↗
PubMed2006

Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity.

Recognition of specific pathogen associated molecular patterns (PAMPs) is mediated primarily by members of the Toll-like receptor (TLR) family. Stimulation through these receptors results in quantitative and qualitative changes in antigen presentation and cellular activation, thereby linking innate and adaptive immunity. Consequently, the incorporation of TLR-ligands into vaccines could result in more potent and efficacious vaccines. To test this hypothesis, we employed a recombinant fusion protein strategy using the TLR5 ligand flagellin fused to specific antigens to promote protective immunity. These purified recombinant fusion proteins demonstrated potent TLR5-specific NF-kappaB dependent activity in vitro. Immunization of mice with the recombinant-flagellin-OVA fusion protein STF2.OVA resulted in potent antigen-specific T and B cell responses that were equal to or better than responses induced by OVA emulsified in Complete Freund's adjuvant. These included rapid and consistent antigen-specific IgG(1) and IgG(2a) antibody responses that were detectable within 7 days of immunization, and the development of protective CD8 T cell responses. Moreover, the enhanced immunogenicity to OVA is dependant on the direct fusion to flagellin, as co-delivery of OVA with flagellin unlinked failed to augment antigen-specific responses in vivo. Similar results were obtained using a recombinant fusion protein comprised of flagellin and a novel polypetide sequence containing two immuno-protective epitopes derived from the Listeria monocytogenes antigens p60 and listeriolysin O. Animals immunized with this recombinant protein demonstrated significant antigen-specific CD8 T cell responses and protection upon challenge with virulent L. monocytogenes. We conclude that immunization with PAMP:antigen fusion proteins induce rapid and potent antigen-specific responses in the absence of supplemental adjuvants. Collectively our data demonstrate that PAMP:antigen fusion proteins offer significant promise for developing recombinant protein vaccines.

Animals↗