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Previous studies have shown that at least three vaccinia virus (VV) late proteins (with apparent molecular asses of 37, 35, and 25 kDa) label with myristic acid. Time course labeling of VV-infected cells with [3H]myristic acid reveals at least three additional putative myristylproteins, with apparent molecular masses of 92, 17, and 14 kDa. The 25-kDa protein has previously been identified as that encoded by the L1R open reading frame, leaving the identities of the remaining proteins to be determined. Sequence analysis led to the preliminary identification of the 37-, 35-, and 17-kDa proteins as G9R, A16L, and E7R, respectively. Using synthetic oligonucleotides and PCR techniques, each of these open reading frames was amplified by using VV DNA as a template and then cloned individually into expression vectors behind T7 promoters. These plasmid constructs were then transcribed in vitro, and the resulting mRNAs were translated in wheat germ extracts and radiolabeled with either [35S]methionine or [3H]myristic acid. Each wild-type polypeptide was labeled with [35S]methionine or [3H]myristic acid in the translation reactions, while mutants containing an alanine in place of glycine at the N terminus were labeled only with [35S]methionine, not with myristic acid. This result provided strong evidence that the open reading frames had been correctly identified and that each protein is myristylated on a glycine residue adjacent to the initiating methionine. Subcellular fractionations of VV-infected cells suggested that A16L and E7R are soluble, in contrast to L1R, which is a membrane-associated protein.  相似文献   

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New vectors for high level expression of recombinant proteins in bacteria.   总被引:26,自引:0,他引:26  
A system has been developed for synthesis and rapid purification of recombinant polypeptides expressed in frame with glutathione S-transferase (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). Expressed fusion proteins are purified from bacterial extracts by glutathione-agarose affinity chromatography. A thrombin protease cleavage site allowed for proteolysis of the fusion protein. We reported the construction of the vector pGEX-KG (K. Guan and J. E. Dixon, 1991, Anal. Biochem. 192, 262-267) which has a glycine-rich "kinker" immediately after the thrombin cleavage site. This kinker dramatically improved the thrombin cleavage efficiency of several fusion proteins. One potential drawback of expressing proteins in this vector is that, following proteolytic cleavage, unrelated amino acids from the vector remain at the amino terminus of the released protein. These extensions could affect enzymatic activity or protein structure. We have constructed two new vectors, pGEX-KT and pGEX-KN, which have the glycine kinker placed N-terminal to the thrombin cleavage site in order to minimize the unrelated amino acids associated with the cleaved protein. The change in location of the kinker had no effect on the increased thrombin cleavage efficiency. A strategy combining the kinker in the vector pGEX-KN with polymerase chain reaction has also been developed to express fusion proteins which when cleaved with thrombin released a protein having no amino terminal extensions of any kind.  相似文献   

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T M Chambers  K Essani  R G Webster 《Gene》1990,95(2):275-278
To assess the utility of two temperature-sensitive (ts) mutant vaccinia viruses as vectors for the conditional in vitro expression of recombinant foreign genes, we have studied the kinetics of expression of foreign genes incorporated into these viruses. At nonpermissive temperature, 40 degrees C, these viruses were defective either in DNA synthesis or in virus assembly. Foreign gene expression was affected by the nature of the ts lesion and by the nature of the vaccinia promoter positioned upstream from the foreign gene. With both vector viruses, a foreign gene controlled by the p7.5 early-late promoter was expressed at both 33 degrees and 40 degrees C. With the DNA synthesis-defective vector virus, foreign gene expression controlled by the p11 DNA synthesis-dependent late promoter was inhibited at 40 degrees C, but could be turned on by shift to 33 degrees C. This ts expression system provides an alternative to use of drugs that inhibit DNA synthesis as a means for experimental manipulation of gene expression. Both vector viruses can be used with existing vaccinia virus expression technology.  相似文献   

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Synthetic DNAs and oligonucleotides, which can be prepared conveniently by combining chemical synthesis and enzymatic methods, have been used extensively in recombinant DNA research. Examples include total gene synthesis, probes for the isolation of specific genes from cDNA or genomic libraries, linkers containing specific restriction sites for cloning, primers for DNA and RNA sequencing, and primers for the construction of specific mutations (either deletion, insertion or point mutations) by oligonucleotide-directed site-specific mutagenesis.This article reviews recent advances in the chemical and enzymatic synthesis of oligo- and polynucleotides and the application of synthetic DNA to the expression of foreign proteins. The synthesis of genes, including structural genes and regulatory genes are reviewed. Oligonucleotide-directed site-specific mutagenesis and use of synthetic DNA to optimize foreign protein expression are also discussed.  相似文献   

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Transactivation of a late herpes simplex virus promoter.   总被引:19,自引:3,他引:16  
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Fatty acid acylation of vaccinia virus proteins.   总被引:1,自引:6,他引:1       下载免费PDF全文
Labeling of vaccinia virus-infected cells with [3H]myristic acid resulted in the incorporation of label into two viral proteins with apparent molecular weights of 35,000 and 25,000 (designated M35 and M25, respectively). M35 and M25 were expressed in infected cells after the onset of viral DNA replication, and both proteins were present in purified intracellular virus particles. Virion localization experiments determined M25 to be a constituent of the virion envelope, while M35 appeared to be peripherally associated with the virion core. M35 and M25 labeled by [3H]myristic acid were stable to treatment with neutral hydroxylamine, suggesting an amide-linked acylation of the proteins. Chromatographic identification of the protein-bound fatty acid moieties liberated after acid methanolysis of M25, isolated from infected cells labeled during a 4-h pulse, resulted in the recovery of 25% of the protein-bound fatty acid as myristate-associated label and 75% as palmitate, indicating that interconversion of myristate to palmitate had occurred during the labeling period. Similar analyses of M25 and M35, isolated from infected cells labeled during a 0.5-h pulse, determined that 46 and 43%, respectively, of the protein-bound label had been elongated to palmitate even during this brief labeling period. In contrast, M25 and M35 isolated from purified intracellular virions labeled continuously during 24 h of growth contained 75 and 70%, respectively, myristate-associated label, suggesting greater stability of these proteins or a favored interaction of the proteins containing myristate with the maturing or intracellular virion.  相似文献   

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P Lee  D E Hruby 《Journal of virology》1993,67(7):4252-4263
The three major vaccinia virus (VV) virion proteins (4a, 4b, and 25K) are proteolytically matured from larger precursors (P4a, P4b, and P25K) during virus assembly. Within the precursors, Ala-Gly-X motifs have been noted at the putative processing sites, with cleavage apparently taking place between the Gly and X residues. To identify the sequence and/or structural parameters which are required to define an efficient cleavage site, a trans-processing assay system has been developed by tagging the carboxy terminus of the P25K polypeptide (precursor of 25K) with an octapeptide FLAG epitope, which can be specifically recognized by a monoclonal antibody. By using transient expression assays with cells coinfected with VV, the proteolytic processing of the chimeric gene product (P25K:FLAG) was monitored by immunoblotting procedures. The relationship between the P25K:FLAG precursor and the 25K:FLAG cleavage product was established by pulse-chase experiments. The in vivo cleavage of P25K:FLAG was inhibited by the drug rifampin, implying that the reaction was utilizing the same pathway as authentic VV core proteins. Moreover, the 25K:FLAG protein was found in association with mature virions in accord with the notion that cleavage occurs concomitantly with virion assembly. Site-directed mutagenesis of the Ala-Gly-Ala motif at residues 31 to 33 of the P25K:FLAG precursor to Ile-Asp-Ile blocked production of the 25K:FLAG product. The efficiency of 25K:FLAG production (33.71%) is, however, approximately only half of the production of 25K (63.98%) within VV-infected cells transfected with pL4R:FLAG. One explanation for the lower efficiency of 25K:FLAG production was suggested by the observation in the immunofluorescent-staining experiment that 25K:FLAG-related proteins were not specifically localized to the virus assembly factories (virosomes) within VV-infected cells, although virosome localization was prominent for P25K-related polypeptides. Since VV core protein proteolytic processing is believed to take place during virion maturation, only the P25K:FLAG which was assembled into immature virions could undergo proteolytic maturation. Furthermore during these experiments, a potential cleavage intermediate (25K') of P25K was identified. Amino acid residues 17 to 19 (Ala-Gly-Ser) of the P25K precursor were implicated as the intermediate cleavage site, since no 25K':FLAG product was produced from a mutant precursor in which the sequence was altered to Ile-Asp-Ile. Taken together, these results provide biochemical and genetic evidence to support the hypothesis that the Ala-Gly-X cleavage motif plays a critical role in VV virion protein proteolytic maturation.  相似文献   

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Galindo I  Lorenzo MM  Blasco R 《BioTechniques》2001,30(3):524-6, 528-9
Vaccinia virus expression vectors are widely used to direct the expression of proteins in eukaryotic cells. Here, we describe a new set of plasmid vectors designed for the expression of histidine-tagged proteins in the vaccinia system. To facilitate the rapid isolation of virus recombinants, the plasmids contain a viral gene (F13L) that serves as an efficient selection marker based on virus plaque phenotype. Histidine codons and restriction sites derived from pET-16b bacterial expression plasmid were included, thus facilitating the transfer of genes between E. coli and vaccinia expression plasmids. Plasmids in which the gene is placed downstream of either a strong vaccinia virus or a T7 promoter were constructed, allowing for constitutive or conditional expression, respectively, of the foreign protein.  相似文献   

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H Tsao  G Q Liu  L Ruan    C M Chu 《Journal of virology》1988,62(12):4832-4834
A bidirectional expression vector containing both the 11KD late promoter (p11) and the presumptive 25KD early promoter (p25) was constructed. These bidirectional vectors have been applied to the expression of hepatitis B surface antigen by using one of the promoters for beta-galactosidase as the marker gene and the other one for hepatitis B surface antigen as the target gene.  相似文献   

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A gene encoding human basic fibroblast growth factor has been chemically synthesized, cloned and expressed in Escherichia coli as a biologically active protein. The 465 bp gene was assembled by enzymatic ligation of 6 pairs of oligonucleotides and cloned in the expression vector pLCII downstream from the strong PL promoter. This promoter directed the synthesis of a fusion protein between a 31 amino acids fragment of the lambda phage cII protein and bFGF. A four amino acid recognition sequence for the site-specific protease fXa was introduced in the plasmid construct and this allowed cleavage of the fusion protein at the boundary between cII and bFGF. bFGF was purified close to homogeneity using a Heparin-Sepharose column and Mono S cation exchange chromatography. The use of the pLCII expression system resulted in the accumulation of 20 to 25 mg of purified bFGF per l of bacterial culture. The recombinant bFGF was mitogenic for mouse 3T3 fibroblasts and the dose-response curve was similar to the one for native bFGF.  相似文献   

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Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10(4) PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10(3) PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy.  相似文献   

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