Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:Q9NRP7 (fused)
 58,367 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have expressed an active recombinant E1 decarboxylase component of the mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli by subcloning mature E1 alpha and E1 beta subunit cDNA sequences into a bacterial expression vector. To permit affinity purification under native conditions, the mature E1 alpha subunit was fused with the affinity ligand E. coli maltose-binding protein (MBP) through an endoprotease Factor Xa-specific linker peptide. When co-expressed, the MBP-E1 alpha fusion and E1 beta subunits were shown to co-purify as a MBP-E1 component that exhibited both E1 activity and binding competence for recombinant branched-chain E2 component. In contrast, in vitro mixing of individually expressed MBP-E1 alpha and E1 beta did not result in assembly or produce E1 activity. Following proteolytic removal of the affinity ligand and linker peptide with Factor Xa, a recombinant E1 species was eluted from a Sephacryl S-300HR sizing column as an enzymatically active 160-kDa species. The latter showed 1:1 subunit stoichiometry, which was consistent with an alpha 2 beta 2 structure. The recovery of this 160-kDa recombinant E1 species (estimated at 0.07% of total lysate protein) was low, with the majority of the recombinant protein lost as insoluble aggregates. Our findings suggest that the concurrent expression of both E1 alpha and E1 beta subunits in the same cellular compartment is important for assembly of both subunits into a functional E1 alpha 2 beta 2 heterotetramer. By using this co-expression system, we also find that the E1 alpha missense mutation (Tyr-393----Asn) characterized in Mennonites with maple syrup urine disease prevents the assembly of soluble E1 heterotetramers.
 ...
PMID:Expression and assembly of a functional E1 component (alpha 2 beta 2) of mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli. 164 40

To localize regions on IgG bound by rheumatoid factors (RF), we studied IgM RF binding to chimeric IgG antibodies consisting of murine V regions fused to human constant regions. Using a modified RF ELISA, we showed that polyclonal RF from rheumatoid arthritis patients bound IgG1, 2, and 4 strongly; IgG3 was also bound, although less well. The majority of 18 monoclonal RF from patients with Waldenstrom's macroglobulinemia bound IgG1, 2, and 4 only. In contrast to RF from RA, 14 of 18 monoclonal RF did not react with IgG3. Only 3 of 18 monoclonal RF bound IgG3 well. By shuffling C region domains between IgG3 and IgG4, we showed that sequence variation in the CH3 domain is responsible for the differential binding of monoclonal RF to IgG3 and IgG4. Hybrid IgG3/IgG4 antibodies containing the CH3 domain of IgG4 were bound by monoclonal RF, whereas those containing the CH3 domain of IgG3 were not. To evaluate the contribution of the N-linked carbohydrate moiety at Asn-297 to RF binding sites on IgG, we measured RF binding to aglycosylated IgG antibodies produced by mutating Asn-297 to another amino acid. Glycosylated and aglycosylated IgG1, 2, and 4 were bound identically by monoclonal and polyclonal RF. Aglycosylated IgG3, however, was bound better than glycosylated IgG3 by polyclonal RF and by IgG3-reactive monoclonal RF.
 ...
PMID:Molecular analysis of IgM rheumatoid factor binding to chimeric IgG. 170 8

Ricin B chain (RTB) is an N-glycosylated galactose-specific lectin which folds into two globular domains. Each domain binds one galactoside. The x-ray crystallographic structure has shown that the two binding sites are structurally similar and contain key binding residues which hydrogen bond to the sugar, and a conserved tripeptide, Asp-Val-Arg. We have used oligonucleotide site-directed mutagenesis to change either the binding residues or the homologous tripeptide in one or other or in both of the sites. The 5' signal sequence and RTB coding region were excised from preproricin cDNA and fused in frame to generate preRTB cDNA. Transcripts synthesized in vitro from wild-type or mutant preRTB cloned into the Xenopus transcription vector pSP64T using SP6 RNA polymerase, were microinjected into Xenopus oocytes. The recombinant products were segregated into the oocyte rough endoplasmic reticulum and core-glycosylated, and the N-terminal signal peptide was removed. Mutating sugar binding sites individually did not abrogate the lectin activity of RTB. When both sites were changed simultaneously, RTB was produced which was soluble and stable but no longer able to bind galactose. Changing the Asn residues of the two RTB N-glycosylation sites to Gln showed that oligosaccharide side chains were essential for both the stability and biological activity of recombinant RTB.
 ...
PMID:Mutational analysis of the galactose binding ability of recombinant ricin B chain. 171 62

The lactose-assimilating yeast, Kluyveromyces lactis, has been developed as a microbial host for the synthesis and secretion of human proteins. Here, we report the use of multi-copy vectors based on the 2 mu-like plasmid pKD1 from Kluyveromyces drosophilarum [Chen et al., Nucleic Acids Res. 14 (1986) 4471-4481] for the secretion of recombinant human interleukin-1 beta (reIL-1 beta). High levels of reIL-1 beta were secreted into the growth medium when the structural gene was fused in-frame to a synthetic secretion signal derived from the 'pre'-region of the K. lactis killer toxin. N-terminal sequencing of the excreted protein showed highly efficient (greater than 95%) maturation of the signal sequence. Synthesis as prepro-IL-1 beta, the 'pro'-sequence being derived from the human serum albumin-encoding gene, resulted in equally efficient secretion of mature IL-1 beta. Cytoplasmic production of Met-IL-1 beta, without a secretion signal, was found to be toxic to K. lactis. As in Saccharomyces cerevisiae [Baldari et al., EMBO J. 6 (1987) 229-234], but unlike native human IL-1 beta, K. lactis reIL-1 beta is glycosylated. This glycosylation led to a 95% loss of its biological activity. Removal of the carbohydrate chains by endo-beta-N-acetyl-glucosamidase H treatment fully restored the biological activity. A modified form of IL-1 beta (Asn7----Gln7), in which the unique site for Asn-linked glycosylation was deleted, exhibited the same biological activity as native IL-1 beta. The level of secretion of mature recombinant IL-1 beta ws glycosylation-independent.
 ...
PMID:High-level secretion of correctly processed recombinant human interleukin-1 beta in Kluyveromyces lactis. 174 98

Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus.
 ...
PMID:Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity. 186 56

A gene encoding a variant (lacking amino acids 6-173) of human tissue-type plasminogen activator (t-PA), consisting only of the second kringle domain (K2) and the serine protease domain (P), was fused to a DNA segment coding for the signal peptide of staphylococcal protein A and a synthetic gene coding for a protein with ability to bind immunoglobulin G (IgG). The fusion protein which was synthesized in Escherichia coli and secreted into the growth medium, was found to be fibrinolytically active. Purification of the fusion protein was performed in a single step by affinity chromatography with immobilized IgG. Enzymatically active K2P was liberated from the fusion protein by cleavage at a unique Asn-Gly dipeptide sequence using hydroxylamine. These results demonstrate that a variant of human t-PA can be synthesized and secreted by E. coli as a fibrinolytically active fusion protein, which upon specific cleavage yields an active variant t-PA of the expected size.
 ...
PMID:Synthesis and secretion of a fibrinolytically active tissue-type plasminogen activator variant in Escherichia coli. 190 33

CD4-IgG is a homodimer of a hybrid polypeptide consisting of the two amino-terminal domains (residues 1-180) of human CD4 fused to the hinge region and the second and third constant-sequence (CH2 and CH3) Fc domains (residues 216-441) of human immunoglobulin G (IgG-1). This antibody-like molecule, termed an immunoadhesin, was produced in an effort to combine the binding specificity of CD4 with several potentially desirable properties of IgG molecules [Capon et al. (1989) Nature 337, 525-531]. The structural characteristics of the molecule have been evaluated to demonstrate that CD4-IgG has the same features as the N-terminal region of soluble CD4, while retaining those expected for the Fc portion of human IgG. Identification of peptides recovered from the tryptic map confirmed 98.8% of the expected structure of CD4-IgG. The detection of glucosamine in peptides containing Asn257 and the retention time shift of this tryptic peptide after deglycosylation confirmed the presence of Asn-linked oligosaccharides at this position. Four pairs of intrachain and two interchain disulfide bonds were also established.
 ...
PMID:Structural characterization of a recombinant CD4-IgG hybrid molecule. 226 86

We describe a cDNA clone derived from mRNA of asexual blood-stages of the malaria parasite Plasmodium falciparum. This clone, designated Ag319, expresses a P.falciparum antigen fused to beta-galactosidase in Escherichia coli. Human antibodies from Papua New Guinea were affinity-purified by adsorption to extracts of Ag319 immobilized on CNBr-Sepharose. The antibodies reacted predominantly with P. falciparum polypeptides of Mr 220,000 and 160,000, and a number of ill-defined lower molecular weight species. Antibodies reacted in indirect immunofluorescence with all asexual blood-stages although the antigen appeared to be most abundance in the schizont. Surprizingly the antibodies also reacted with sporozoites. The amino acid sequence predicted from the complete nucleotide sequence of this clone is remarkable because 40% of the residues are Asn, and so the antigen has been termed the Asparagine-Rich Protein (ARP). Like other P. falciparum antigens, ARP contains tandemly repetitive sequences, based on the tetrapeptide Asn-Asn-Asn-Met and we have confirmed that these represent natural epitopes by reaction of the corresponding synthetic peptides with human antibodies. Surprisingly, ARP is also rich in Asn outside the tandem repeats.
 ...
PMID:An asparagine-rich protein from blood stages of Plasmodium falciparum shares determinants with sporozoites. 242 Dec 57

A cDNA fragment coding for poliovirus capsid polypeptide VP1 was inserted into a simian virus 40 (SV40) genome in the place of the SV40 VP1 gene and fused in phase to the 3' end of the VP2-VP3 genes. Simian cells were infected with the resulting hybrid virus in the presence of an early SV40 mutant used as a helper. Indirect immunofluorescence analysis of the infected cells using anti-poliovirus VP1 immune serum revealed that the SV40/poliovirus fusion protein was located inside the cell nucleus. Deletions of various lengths were generated in the SV40 VP2-VP3 portion of the hybrid gene using BAL31 nuclease. The resulting virus genomes expressed spliced fusion proteins whose intracellular location was either intranuclear or intracytoplasmic, depending on the presence or absence of VP2 amino acid residues 317 to 323 (Pro-Asn-Lys-Lys-Lys-Arg-Lys). This was confirmed by site-directed mutagenesis of the Lys residue at position 320. Modification of Lys-320 into either Thr or Asn abolished the nuclear accumulation of the fusion protein. It is concluded that at least part of the sequence of VP2 amino acids 317 to 323 allows VP2 and VP3 to remain stably located inside the cell nucleus. The proteins are most probably transported from the cell cytoplasm to the cell nucleus by interaction, with VP1 acting as a carrier.
 ...
PMID:The intranuclear location of simian virus 40 polypeptides VP2 and VP3 depends on a specific amino acid sequence. 282 20

We have mapped a sequence determinant in the vacuolar glycoprotein carboxypeptidase Y (CPY) that directs intracellular sorting of this enzyme. Through the study of hybrid proteins, consisting of amino-terminal segments of CPY fused to the secretory enzyme invertase, we have found that the N-terminal 50 amino acids of CPY are sufficient to direct delivery of a CPY-Inv hybrid protein to the yeast vacuole. Our data suggest that this 50 amino acid segment of CPY contains two distinct functional domains; an N-terminal signal peptide followed by a segment of 30 amino acids that contains the vacuolar sorting signal. Deletion of this putative vacuole sorting signal from an otherwise wild-type CPY protein leads to missorting of CPY. Furthermore, examination of the Asn-linked oligosaccharides present on CPY and CPY-Inv hybrid proteins suggests that an additional determinant in CPY specifies the extent to which these proteins are glycosylated in the Golgi complex.
 ...
PMID:Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease. 302 48


1 2 3 4 5 6 7 8 9 Next >>