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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

HIV-1 reverse transcriptase from the HIV-1 strain WMF 1.13 was expressed in Escherichia coli JM 105 using a pKK233-2 vector. The bacteria were cultivated in a 20-l fermentor with 14-l net volume using M9ZB medium containing bactotryptone and yeast extract. After induction of reverse transcriptase (RT) expression by addition of isopropyl-beta-D-thiogalactopyranoside the enzyme concentration was monitored. Both soluble and inclusion-body deposited RT were detected by Western blots. Inclusion-body formation was confirmed by transmission electron microscopy. Further purification of soluble and insoluble RT was investigated. After cell desintegration by enzymatic treatment combined with osmotic shock and centrifugation, the supernatant was desalted by size-exclusion chromatography and further purified by DEAE-Sepharose FF, AF-Heparin Toyopearl 650 M and Fractogel EMD TMAE 650 (S). The results of the purification steps were monitored by SDS-PAGE with silver staining, non-radioactive RT assay and protein determination with Coomassie Blue. The sediment was extracted with 6 M GuHCl and after clarification and conventional refolding, treated in the same manner as soluble RT. This method is well suited for studying fermentation conditions as well as purification conditions. The RT is expressed in approximately equal amounts as soluble and insoluble enzyme.
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PMID:Laboratory-scale production and purification of recombinant HIV-1 reverse transcriptase. 753 52

The human immunodeficiency virus type 2 (HIV-2) Nef protein expressed in Escherichia coli forms highly stable homooligomeric complexes in vitro. Similarly, the native protein synthesized in the persistently infected H9 T cell line also forms stable homooligomers in vivo. To determine whether homooligomer formation is mediated by the leucine zipper-type sequence located in the middle region of the protein, site-directed mutagenesis was used to introduce double and triple point mutations at heptad leucine positions L1, L2, and L4 within the HIV-2NIHZ Nef protein sequence. Here, we show that substitution of a serine residue for the L1 (residue 108) and L2 (residue 115) heptad leucines, and a glutamine residue for the L4 (residue 129) heptad leucine, did not prevent Nef homooligomer formation in vitro. However, a more drastic substitution of alpha-helix-breaking proline residue for the L2 and L4 heptad leucines significantly abrogated ability of the protein to form stable homooligomers. In addition, because significantly higher levels of the Nef oligomers were consistently observed under the nonreducing SDS-PAGE condition, site-specific mutagenesis was also used to examine the role of cysteine residues in generating disulfide-linked Nef dimers in vitro. Here, we also show that single cysteine-to-glycine substitutions at positions 28, 32, or 55 drastically reduced covalent Nef dimer formation and thermal stability of the Nef protein in vitro. Therefore, these results demonstrate that the leucine zipper-type motif in the HIV-2 Nef protein mediates stable homooligomer formation in vitro, and also establish a role for covalent disulfide bonds in the formation of linked Nef dimers and thermal stability of the monomer Nef in vitro.
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PMID:Oligomerization of the HIV type 2 Nef protein: mutational analysis of the heptad leucine repeat motif and cysteine residues. 773 98

Poliovirus RNA polymerase (3Dpol) was cross-linked to [32P]ribonucleoside triphosphates (NTPs) by reduction of oxidized NTP-protein complexes. Cross-linked complexes were digested with cyanogen bromide, and resulting peptides were fractionated by reverse-phase HPLC. 32P-Labeled peptides were purified by secondary HPLC fractionation and/or additional digestion with endoproteinases Glu-C, TPCK-trypsin, or Asp-N followed by another HPLC fractionation. N-Terminal sequences of the major [32P]-peptides were determined, and approximate sizes of these peptides were obtained by SDS-polyacrylamide gel electrophoresis. Two major NTP binding sites in 3Dpol were found. One site was between Asp-266 and Met-286; possible binding residues in this fragment were Lys-276, Lys-278, or Lys-283. A second binding site was between Ala-57 and Met-74 with Lys-61 or Lys-66 as possible binding residues. Alignment of these regions on the known structure of HIV-1 reverse transcriptase allowed us to predict the position of the downstream nucleotide binding site in the conserved "fingers" subdomain present near the active site cleft of both RNA and DNA polymerases. The N-terminal nucleotide binding site is not contained within a region that is conserved among other polymerases.
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PMID:Identification of nucleotide binding sites in the poliovirus RNA polymerase. 775 55

Glycosylation is necessary for HIV-1 gp120 to attain a functional conformation, and individual N-linked glycans of gp120 are important, but not essential, for replication of HIV-1 in cell culture. We have constructed a mutant HIV-1 infectious clone lacking a signal for N-linked glycosylation in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by ConA and a conformation dependent human antibody IAM-2G12. This suggests that the N-linked glycan in the V1-loop modulates the three-dimensional conformation of gp120, without changing the overall functional integrity of the molecule.
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PMID:Identification of an N-linked glycan in the V1-loop of HIV-1 gp120 influencing neutralization by anti-V3 antibodies and soluble CD4. 783 33

Selective glycosylation of "prompt" stress glycoproteins (P-SG), mainly P-SG67 and P-SG64 (M(r) of 64,000, pI = 5.1), occurs immediately during acute heat-stress. In the present study, P-SG64 was purified by sequential gel filtration, anion-exchange, affinity chromatography, and two-dimensional isoelectric focusing/SDS-PAGE. Purified P-SG64 was further characterized by microsequencing of a peptide fragment, PT-61, which showed a 100% sequence homology with calreticulin, suggesting that P-SG64 is identical to calreticulin. PT-61 also showed 55%, 58% and 63% sequence homologies with calnexin, HIV-1 gp120 and HIV-2 envelope polyprotein, respectively. 45Ca2+ overlay studies confirmed Ca(2+)-binding of P-SG64. P-SG67 was also recently identified as calreticulin (8), which suggests that CHO cells either have two isoforms of calreticulin or express variable states of calreticulin glycosylation during acute heat stress. The role of intracellular Ca2+ ([Ca2+]i) during heat-induced "prompt" glycosylation was also examined and indicated an 8-fold increase in [Ca2+]i. Chelation of this increased cytoplasmic Ca2+ by BAPTA reduced glycosylation of P-SG67/P-SG64/calreticulin only by approximately 20%. This observation suggests that altered [Ca2+]i homeostasis is not directly linked to calreticulin glycosylation, instead, heat-induced calreticulin glycosylation is a Ca(2+)-independent effect.
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PMID:Prompt glycosylation of calreticulin is independent of Ca2+ homeostasis. 799 12

Based on our findings that HIV-1 gp41 can bind independently of CD4 to the human T cell line H9, B cell line Raji and monocytic cell line U937, as well as human peripheral blood mononuclear cells preferentially to B lymphocytes and monocytes, we examined whether soluble protein for HIV-1 gp41 binding exists in the cell culture supernatant of Raji and U937. Using HIV-1 recombinant soluble gp41 (sgp41; Env amino acid 539-684) attached to sepharose beads, the cell culture supernatants of Raji and U937 were absorbed. By SDS-PAGE of sgp41-eluates of these cell culture supernatants three protein bands of 70, 75 and 92kD were stained with Coomassie blue. By Western blot (ligand blot) analysis using sgp41, two protein bands of 70 and 75kD were observed in sgp41-eluates from Raji and U937 cell culture supernatants. These sgp41-eluates could inhibit the sgp41 binding to Raji and U937 cells. Our data indicate that Raji and U937 cells produce two soluble binding proteins for HIV-1 gp41.
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PMID:HIV-1 gp41 binds to two proteins in cell culture supernatant of human B cell line Raji and monocyte cell line U937. 808 54

The gene encoding the major envelope glycoprotein of the HIV-SF2 isolate was engineered for the secretion of recombinant gp120 (rgp120SF2) from permanent Chinese hamster ovary (CHO) cell lines. Cellular production methods were scaled up and a method for purification of the secreted glycoprotein was devised. Mild purification conditions were selected in order to preserve the native structure of the protein. rgp120SF2 exhibits a molecular weight of 120 kDa in reduced or nonreduced SDS gels; thus the polypeptide chain is intact. Deglycosylated rgp120SF2 has the predicted molecular weight of the polypeptide backbone, 54 kDa. Gel-filtration HPLC in a nondenaturing buffer at neutral pH yields a molecular weight estimate of approximately 120 kDa. Purified rgp120 closely resembles authentic viral gp120 by several physical, chemical, and immunochemical tests. rgp120SF2 reacts strongly with human HIV-positive sera, monoclonal antibodies reactive with HIV-SF2 and HIV-MN viral envelope, and a human virus-neutralizing monoclonal antibody that maps to a conserved discontinuous epitope on HIV-1 gp120. Purified rgp120SF2 forms a 1:1 molecular complex with soluble recombinant human CD4 (rCD4) receptor, as demonstrated by gel-filtration HPLC; binding is high affinity (Kd approximately 2 x 10(-9) M).
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PMID:Nonaffinity purification of recombinant gp120 for use in AIDS vaccine development. 814 40

The negative factor, Nef, of HIV-1 was found to associate to an extent of 16-42% with the detergent insoluble cytoskeletal fraction of T lymphocytes. Furthermore, Escherichia coli expressed Nef protein was found to bind during in vitro reactions with the cytoskeletal matrix to an extent of 30-50%. Cytoskeletal association of Nef was significantly enhanced by myristoylation. The specificity of the myristoylation-enhanced binding was demonstrated by the lack of an effect of myristoylation on binding of the HIV-1 Gag protein to the cytoskeleton. Cytoskeletal binding was saturable, and inhibited by high concentrations of sodium chloride, or with SDS or urea. Binding of Nef to the cytoskeletal matrix may be important in mediating its effects on HIV-1 replication.
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PMID:Myristoylation-enhanced binding of the HIV-1 Nef protein to T cell skeletal matrix. 821 77

Based on our findings that HIV-1 gp41 independently of CD4 can bind to the helper T cell line H9 and B cell line Raji, we characterized the putative binding of HIV-1 gp41 to the monocyte cell lines U937 and HL60. Using flow cytometry (FACS) we examined the binding of soluble gp41 (sgp41; env amino acids 539-684) to these monocyte cell lines. Using sgp41 attached to Sepharose beads, U937 cell lysates were absorbed. The sgp41 eluate of U937 cell lysates could inhibit sgp41 binding to U937 cells. With SDS-PAGE of sgp41 eluate of U937 cell lysates, three strong protein bands, (37, 45 and 62 kDa) and two weak bands (49 and 92 kDa) were stained with Coomassie blue. With Western blot (ligand blot) analysis using sgp41, three strong protein bands (37, 49 and 62 kDa) and a very weak band (42-45 kDa) were observed in sgp41 eluate of U937 cell lysates. The results suggest that the four proteins 37, 42-45, 49 and 62 kDa in U937 cell lysates are possible candidates for the putative gp41 receptor(s). We compared the blocking activities of sgp41 eluates from different cell lysates. Not only U937 and Raji lysate-sgp41 eluates, but also H9 and HL60 lysate-sgp41 eluates could block sgp41 binding to U937 and Raji cells. The results indicate that the sgp41-binding proteins on U937, or Raji (H9 and HL60, respectively) probably could have an identical blocking (or binding) specificity; these cell types carry very similar receptor(s) for HIV-1 gp41 binding.
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PMID:The human monocyte cell line U937 binds HIV-1 gp41 by proteins of 37, 45, 49, 62 and 92 kDa. 822 5

A large-scale immunoaffinity (IA) purification process was developed for the isolation of recombinant soluble antigen CD4 (sCD4) from Escherichia coli fermentations. The monoclonal antibody used for IA purification of sCD4 recognized a conformation-dependent epitope on the surface of domain 1 of CD4. IA chromatography was used to purify both sCD4-183, consisting of the N-terminal 183 amino acids of human CD4, and sCD4-PE40, a fusion protein consisting of the N-terminal 178 amino acids of CD4 and amino acids 1-3 and 253-613 of Pseudomonas exotoxin A (PE40). sCD4-183 was purified from E. coli cell pellets using cell disruption, protein solubilization, oxidation, Q-Sepharose anion-exchange and IA chromatography steps. sCD4-PE40 was purified from cell pellets using cell disruption, protein solubilization, oxidation, Cu(2+)-immobilized metal-affinity chromatography, anion-exchange and IA chromatography steps. The IA-purified sCD4 analogues demonstrated the correct apparent molecular masses on SDS/PAGE. The immobilized monoclonal antibody appeared to select for correctly folded CD4 protein, since sCD4-183 and sCD4-PE40 purified by the IA method bound human-immunodeficiency-virus glycoprotein gp120 (HIV gp120) in vitro. sCD4-PE40 purified by IA chromatography also inhibited protein synthesis in CV-1 cells expressing HIV gp120/160 at the cell surface. Relatively high recoveries of sCD4-183 and sCD4-PE40 were observed in the IA step of the purification process (71 and 79% recovery respectively). The results demonstrate that immobilized monoclonal antibodies directed against conformational epitopes may be used for rapid purification of gram amounts of correctly folded protein from mixtures of oxidized E. coli proteins.
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PMID:Large-scale immunoaffinity purification of recombinant soluble human antigen CD4 from Escherichia coli cells. 829 11


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