Expression of the gag gene of human T-cell leukemia virus type I in Escherichia coli and its diagnostic use

An expression plasmid, pHY202, was constructed which directs the synthesis of a fusion protein encoded by the gag sequence of human T-cell leukemia virus type I (HTLV-I) inserted into the lacZ' gene. Escherichia coli cells harboring pHY202 produced the 43-kDal LacZ'-Gag fusion protein with a yield of approx. 0.3% of total soluble proteins. The fusion protein is specifically recognized by monoclonal antibodies against the Gag proteins p19 and p24, and could be applicable for the diagnosis of HTLV-I infection, because almost all sera from HTLV-I carriers gave a positive response in the enzyme-linked immunosorbent assay (ELISA) employing the LacZ'-Gag hybrid protein purified by immunoaffinity column chromatography.     

Expression of the murine leukemia virus protease in fusion with maltose-binding protein in Escherichia coli

The protease of murine leukemia virus (MLV) was cloned into pMal-c2 vector, expressed in fusion with maltose-binding protein (MBP), and purified to homogeneity after Factor Xa cleavage of the chimeric protein. Substantial degradation of the fusion protein was observed during expression, which severely diminished the yield. The degree of degradation of the fusion protein was even more pronounced when a single-chain form of the MLV protease was cloned after the gene coding for MBP. To increase the yield, a hexahistidine tag with an additional Factor Xa cleavage site was cloned after the protease and nickel chelate affinity chromatography was used as the first purification step. The modified procedure resulted in substantially higher yield as compared to the original procedure. The degradation of hexahistidine-tagged active site mutant MLV protease was very low and comparable to that obtained with hexahistidine-tagged MBP, but purified MLV protease alone was not able to degrade purified MBP, suggesting that during expression the active MLV protease may activate bacterial proteases which appear to be responsible for the degradation of the fusion proteins.     

The bovine leukemia virus p34 is a transactivator protein.

Recombinant Moloney murine retroviruses containing the BLV post-envelope long open reading frame were constructed and transfected into the psi 2 packaging cell line. They were shown to encode and to express a 34-kd protein able to transactivate the BLV long terminal repeat-directed gene expression in the respective transfected cells. These data demonstrate that the BLV X-LOR gene encodes a p34 transactivator product. Furthermore, the different cell lines produced infectious recombinant retroviruses capable of transferring X-LOR genes into recipient cells. The availability of the BLV transactivator protein should allow us to understand the role of the transactivator protein in BLV-induced leukemogenesis.     

Cloning of the bovine leukemia virus proteinase in Escherichia coli and comparison of its specificity to that of human T-cell leukemia virus proteinase

The proteinase of bovine leukemia virus (BLV) was cloned into pMal-c2 vector with N-terminal or with N- as well as C-terminal flanking sequences, and expressed in fusion with maltose binding protein. The proteinase self-processed itself from the fusion protein during expression and formed inclusion bodies. The enzyme was purified from inclusion bodies by cation-exchange chromatography followed by gel filtration. Specificity of the enzyme was compared to that of human T-cell leukemia proteinase type 1. Although the two viruses belong to the same subfamily of retroviruses, the differences in their proteinase specificity, based on kinetics with oligopeptide substrates representing naturally occurring cleavage sites as well as on inhibition pattern, appear to be pronounced.     

Expression of human T-cell leukemia virus type I protease in Escherichia coli.

Human T-cell leukemia virus type I (HTLV-I) genome is believed to encode its own protease, although the protease has not yet been detected. To identify the HTLV-I protease, an in-frame gag (3' portion)-prt region was expressed in Escherichia coli. The 14-kDa product was detected using antisera against a synthetic peptide mimicking the fragment of HTLV-I protease, although the molecular weight of the primary translational product was 27,000. A cell extract had a proteolytic activity to cleave a synthetic peptide substrate containing the cleavage site of gag p19/p24 at the correct site in vitro. Replacement of the putative active site Asp-64 with Gly abolished both in vivo processing activity and in vitro proteolytic activity. These results suggest that the 14-kDa product is the mature enzymatically active HTLV-I protease generated through posttranslational autoprocessing in E. coli.     

Expression of the x-lor gene of human T-cell leukemia virus I in Escherichia coli.

The 3'-terminal regions of the human T-cell leukemia virus I (HTLV-I) and HTLV-II genomes encode a novel gene product. We showed that expression of this region fused to the beta-galactosidase gene in bacteria produces a protein recognized by adult T-cell leukemia-lymphoma patient sera. Rabbit antibodies raised against this protein specifically precipitated the 42-kilodalton x-lor gene protein from HTLV-I-infected cells.     

Expression of fatty acid-binding protein from bovine heart in Escherichia coli

Summary The coding part of the cDNA of cardiac fatty acid-binding protein (cFABP) from bovine heart was cloned into the vector pKK233-2. After induction with isopropyl--d-thiogalactopyranoside cFABP was found in a soluble form in the cytosol of plasmid transformed E. coli amounting up to 5.7% of the soluble protein. cFABP was detected after SDS-polyacrylamide gelelectrophoresis and/or isoelectric focusing and Western blot by immuno-staining and was determined quantitatively by a solid phase enzyme-linked immuno sorbent assay. The cFABP produced by bacteria binds oleic acid with high affinity as shown by comigration of protein and ligand in both gelfiltration and isoelectric focusing. cFABP was purified from bacterial lysates to near homogeneity and resolved into four isoproteins.     

Isolation of a bovine plasma fibronectin-containing complex which inhibits the expression of bovine leukemia virus p24

Bovine leukemia virus (BLV) is a deltaretrovirus that infects cattle worldwide. In agriculturally intensive regions, approximately 30% of dairy cows are BLV infected. Like the human T-cell leukemia virus (HTLV), there is a lengthy period of viral quiescence after initial infection with BLV. Unlike HTLV, BLV resides predominantly in B cells. Lymphoma is observed in less than 10% of BLV-infected adult cattle. Although viremia is undetectable in vivo, BLV-infected peripheral blood mononuclear cells readily become productive when cultured in vitro. Productivity is markedly diminished when cultures are supplemented with bovine plasma. This inhibitory activity of bovine plasma has been attributed to the "plasma blocking factor" (PBF). Here, we describe the purification of a PBF whose activity was resistant to heating to 65 degrees C for 10 min and was attributable to a fibronectin-containing complex of approximately 320 kDa under nonreducing conditions. By use of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight (mass spectrometry), a protein with a size of 220 kDa and a pI of 5.4 was identified as a member of the fibronectin group of molecules. Both the purified protein and the commercially available bovine fibronectin inhibited BLV production in naturally infected peripheral blood mononuclear cells, although the fibronectin was less biologically active.     

A serological screening assay of human immunodeficiency virus type 1 antibodies based on recombinant protein p24-gp41 as a fusion protein expressed in Escherichia coli

The objective of this study was expression of a recombinant fusion protein p24-gp41 to gain a proper folding pattern of the proteins which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1) for development of a reliable serodiagnostic kit. Serodiagnostic method using enzyme-linked immunosorbent assay (ELISA) with the expressed recombinant fusion protein p24-gp41 was carried out to test the sensitivity and specificity of the protein using human sera and various reference panels from Boston Biomedica Inc. (BBI). The level of the expression was determined to be 30% and the final recovery from fermentation and purification process was calculated as 80 mg/L with more than 98% purity. The developed ELISA assay was demonstrated to have 100 and 99.5% sensitivity and specificity, respectively, detecting anti-HIV-1 antibody using 900 positive and 10,000 negative human sera. The developed assay showed reliable results in comparison with other reference HIV ELISA kits using various BBI panels as well. In conclusion, the recombinant fusion protein p24-gp41 was expressed and used to develop a serodiagnostic kit for screening of the HIV-1 with high sensitivity (100%) and specificity (99.5%) which could be useful for screening large groups of blood donors.     

Expression of bovine inhibin beta subunit in Escherichia coli.

1. A DNA fragment encoding the beta subunit of bovine inhibin was amplified using the polymerase chain reaction and was cloned in plasmids pUC8 and pUR291. 2. Cultures of Escherichia coli TG2 harbouring pKDK37, a pUR291-derived recombinant plasmid, produced a novel protein with a molecular weight of 130,000 corresponding to a beta-galactosidase-inhibin beta fusion protein. 3. The fusion protein was purified from inclusion bodies by solubilization in 8 M urea followed by an ion-exchange and gel permeation chromatography. 4. Analysis by immunoblotting and competitive radioimmuno assay revealed that the fusion protein was recognized by a monoclonal antibody raised against a chemically synthesized peptide for amino acid residues from +82 to +114 of the beta subunit of the bovine inhibin thereby confirming its identity.     

Expression in Escherichia coli of an immunoreactive visna virus transactivating protein

We have cloned the gene encoding the visna 1514 transactivating protein, Tat, into the Escherichia coli lambda pR expression plasmid, pRIT2T. Efficient synthesis of the protein A::Tat fusion protein was obtained in host strains which carried either wild-type or temperature-sensitive (ts) lambda repressors. However, constitutive synthesis of the fusion protein in these host strains resulted in selection against plasmids which synthesized the fusion protein. Efficient repression of the lambda pR promoter was obtained using a ts repressor gene carried on a multicopy plasmid. Synthesis of the fusion protein in this strain was efficient on induction, and reproducible after subculture. Antisera generated against the termini of visna Tat were used to demonstrate that the fusion protein retained the antigenicity of both the N and C termini of the transactivating protein.     

An improved syncytia infectivity assay for the bovine leukemia virus.

Several factors that influence the sensitivity of the syncytia infectivity assay for the bovine leukemia virus (BLV) and BLV-infected lymphocytes have been examined. The use of early-passage indicator bovine embryonic spleen (BESP) cells and their pretreatment with diethylamino-ethyl-dextran (DEAE-D) was essential for optimal sensitivity. Polybrene was less effective than DEAE-D. The combination of DEAE-D and polybrene was more effective than DEAE-D alone when BLV-infected leukocytes were used as the inoculum, but not when the inoculum was a cell-free BLV preparation. Using BESP cell passages 4 to 11 as indicators, reproducible titers were obtained when aliquots of the same virus stock were assayed at different times after freezer storage. When assaying peripheral blood lymphocytes from infected cattle, optimal syncytia responses were observed consistently by inoculating 5 X 10(6) viable lymphocytes per 60-mm Falcon dish. Centrifugation of peripheral blood leukocytes from BLV-infected cattle in discontinuous bovine serum albumin gradients can be used to separate a subpopulation of infected lymphocytes. Use of this subpopulation as the inoculum, rather than unseparated buffy-coat leukocytes, greatly increases the sensitivity of the syncytia infectivity assay.     

Syncytia infectivity of assay of bovine leukemia virus

Bovine embryonic spleen cell cultures were examined to find several factors influencing the specificity, sensitivity and reproducibility of the syncytia infectivity assay of bovine leukemia virus (BLV). The highest sensitivity of the assay were observed when cell sheets of 30 to 50% confluence were inoculated with a stock of BLV, and when cells containing 4 or more nuclei were counted as syncytial cells. Treatment of the cell sheets with a diethylamino-ethyl-dextran solution (25 micrograms/ml) prior to BLV inoculation was found to be essential for the optimal induction of syncytia. Low-passage cultures were found to be more susceptible to the induction of syncytia by BLV than high-passage cultures. Cell-free BLV preparations decreased in syncytia-inducing ability to some extent by the first cycle of freezing (at -70 degrees C) and thawing. No further decrease, however, was caused by repeated cycles of freezing and thawing or by prolonged incuvation at -80 degrees C. The syncytia-inducing activity of BLV was inhibited by all the BLV-precipitating antibody-positive sera originated from both cases of the adult form of bovine leukosis and cases of persistent lymphocytosis. It was not inhibited by the sera of 16 of 17 cattle apparently healthy and negative for BLV-precipitating antibody. These results indicate that the syncytia infectivity assay and syncytia inhibition test are specific for BLV.     

牛呼吸道合胞体病毒G蛋白的截短表达与鉴定

经生物学软件DNA Star分析,将牛呼吸道合胞体病毒G基因截短成2个片段G1和G2。然后用人工合成的牛呼吸道合胞体病毒G基因为模板,用PCR分别扩增G1和G2基因片段,其大小分别为570 bp和308 bp。将目的片段定向克隆到pET30a表达载体中,酶切及测序鉴定均正确后,转化BL21表达菌,经IPTG诱导后G1和G2基因片段都获得了表达,且都为可溶性表达。用Ni柱亲和层析法在非变性条件下纯化重组蛋白,经免疫印迹试验鉴定证明纯化的重组蛋白G1具有良好的抗原性和特异性,而重组蛋白G2无反应性。应用纯化的重组蛋白G1进行的间接ELISA与免疫印迹试验在国内牛血清中检测到了BRSV血清抗体。本研究所表达的重组蛋白G1为基于牛呼吸道合胞体病毒G蛋白的血清学诊断方法的建立与牛呼吸道合胞体病毒G蛋白生物学功能的研究奠定了基础。     

Expression of bovine mitochondrial tRNASer GCU derivatives in Escherichia coli.

By replacing a stretch of five A-U base pairs in the acceptor stem with G-C pairs, mitochondrial tRNA-SerGCU lacking a D arm could be expressed in Escherichia coli cells in considerable amounts. The expressed tRNA with no modified nucleoside was serylated in vitro with the mitochondrial enzyme. The tRNASerGCU derivatives carrying identity elements for alanine tRNA and the related anticodons were expressed. However, this expression event did not affect cell growth, probably because the expression started from the late log phase, which suggests that these mitochondrial tRNA derivatives are not involved in E.coli gene expression systems. Although there are some restrictions in the secondary structure of tRNAs that can be expressed by this method, it could prove useful for preparing large amounts of heterologous tRNAs in vivo.     

Expression of a proline-enriched protein in Escherichia coli.

The feasibility of expressing repeated synthetic codons in bacterial cells was demonstrated by showing that repeated codons for proline were expressed in Escherichia coli. Recombinant DNA technology was used to clone synthetic polydeoxyguanylate:polydeoxycytidylate into the PstI site of plasmid pBR322. Recombinant plasmid pGC139 was shown by means of HaeIII restriction digestion to contain approximately 41 cloned base pairs; the cloned sequence was expressed as a fusion to an ampicillinase protein. The resulting protein, enriched in proline, was expressed from plasmid pGC139 in E. coli maxicells. Extension of this technology could lead to improvement in the production of amino acids and to nutritional enrichment of single-cell protein.     

Expression of the bovine leukemia virus X region in virus-infected cells.

Bovine leukemia virus, like its closest relatives the human T-cell leukemia virus types I and II, contains a 1.8-kilobase X region between the env gene and the 3' long terminal repeat. In this communication, we report the detection and characterization of a subgenomic mRNA from which this X region is presumably translated. This mRNA was produced by a complex splicing mechanism which resulted in juxtaposition of the 5' end of the env gene and the two overlapping X-region open reading frames. Translation of this mRNA could yield at least two distinct proteins depending on which initiation codon is used. Detection of the protein encoded by the BLV X-region long open reading frame has been reported (N. Sagata, J. Tsuzuku-Kawamura, M. Nagayoshi-Aida, F. Shimizu, K.-I. Imagawa, and Y. Ikawa, Proc. Natl. Acad. Sci. USA 82:7879-7883, 1985). Using synthetic peptide antisera, we detected a protein encoded by the short open reading frame in virus-infected cells. The protein migrated in sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 19,000. It is a nuclear phosphoprotein.