Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome

An RP-HPLC assay was developed for a recombinant adenovirus type 5. During chromatography, intact adenovirus dissociated into its structural components (DNA and proteins) and the viral proteome was separated yielding a characteristic fingerprint. The individual components were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, N-terminal sequencing and amino acid composition. The assay was utilized to measure adenovirus particle concentration through quantification of structural proteins. Each structural protein provided independent measurement of virus concentration allowing verification of accuracy. The assay sensitivity is at or below 2·10⁸ particles. Contrary to the benchmark spectrophotometric assay, the RP-HPLC assay was shown to be insensitive to contaminants common for partially purified adenovirus preparations.     

Biochemical studies on bovine adenovirus type 3. I. Purification and properties.

Bovine adenovirus type 3 (BAV3) was purified and its properties were studied. On productive infection of CKT1 cells (a cell line derived from calf kidney) with BAV3, it was observed that viral DNA synthesis was initiated after about 24 h and its rate was maximal after about 40 h. Maturation of the virus occurred several hours after this. Purified BAV3 was separated into four discrete bands by CsCl density gradient centrifugation (complete, incomplete, empty, and degraded viruses). The complete BAV3 was similar in size and structure to human and avian adenoviruses. Polyacrylamide gel electrophoresis showed that the complete BAV3 virion contained at least 10 polypeptides. The total structural proteins of the virion had a similar amino acid composition to those of human adenoviruses. DNA of the complete virus was a linear duplex and its contour length was 12.3 +/- 0.9 mum. The So20,w value of the DNA was 32.9S and its buoyant density in CsCl was 1.717 g/ml. There was about 25% homology between the DNAs of BAV3 and human adenovirus type 5 by filter hybridization. It was also noted that BAV3 produced incomplete virus. The incomplete virus was similar in morphology to the complete virus and contained almost all the structural polypeptides of the latter, but lacked infectivity. However, its DNA had a deletion(s) (13%) which seemed to locate near a terminal.     

Isolation and characterization of fatty acid binding proteins from mammary tissue of lactating rats.

A protein fraction with fatty acid binding activity has been isolated from mammary tissue from lactating rats by a process involving DEAE-cellulose ion-exchange chromatography, heat treatment, CM-cellulose ion-exchange chromatography and finally ammonium sulphate precipitation. The purified fraction migrated as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 14400. However, when this protein fraction was electrophoresed under non-dissociating conditions, two species were observed in a 4:1 ratio. The two components were separated using h.p.l.c. Both bind fatty acids and appear to have similar amino acid compositions although exhibiting different pI values of 4.8 and 4.9. The mammary fatty acid binding proteins appear to be very similar to the fatty acid binding protein isolated from rat heart based on the electrophoretic mobilities and amino acid composition. The major mammary form (pI 4.9) has been partially sequenced and the amino acid sequences obtained can be aligned with 67 residues of the revised rat heart amino acid sequence [Heuckeroth, Birkenmeier, Levin & Gordon (1987) J. Biol. Chem. 262, 9709-9717]. Both mammary species also showed immunochemical identity to rat heart fatty acid binding protein when tested with an anti-serum raised against the heart protein. Anti-sera raised against the minor mammary form (pI 4.8) specifically precipitated this form under non-denaturing conditions but both forms after they had been denatured. Quantitative immunoassays using the anti-(heart fatty acid binding protein) serum showed that concentrations of the fatty acid binding proteins present in mammary cytosols increase during lactation and increase further after feeding a high-fat diet.     

Characterization and binding properties of human fetal lung fatty acid-binding proteins

When delipidated Mr>10,000 cut-off human fetal lung cytosol was separated on gel filtration and ion-exchange chromatography on Auto-FPLC system, two fatty acid-binding proteins (FABPs) of pI 6.9 and pI 5.4 were purified to homogeneity. On Western blotting analysis with the anti-human fetal lung pI 6.9 FABP, these two proteins showed immunochemical cross reactivity with each other and with purified hepatic FABPs but not with cardiac or gut FABP. These two FABPs have identical molecular mass of 15.2 kDa, which is slightly higher than that of the hepatic proteins (14.2 kDa). Carbohydrate covalently linked to FABPs, that may substantially add to the molecular mass, was not detected in the purified protein preparations. Amino acid analysis revealed that both the proteins have same amino acid composition each containing one Trp residue that is lacking in hepatic FABP. Different isoforms of lung FABP exhibited different binding ability for their natural ligands. These proteins bind palmitoyl CoA with higher affinity than oleic acid. pI 6.9 FABP can more rapidly and efficiently transfer fatty acid than can pI 5.4 FABP from unilammelar liposomes. Thus these FABPs may play a critical role in fatty acid transport during human fetal lung development.Abbreviations AO anthroyloxy - 12-AS 12-(9-anthroyloxy)stearic acid - FABP fatty acid-binding protein - NBD-PE [N-(4-nitrobenzo-2-oxa-1,3-diazole)phosphatidylethanolamine - Pal-CoA palmitoyl coenzyme A - PITC phenylisothiocyanate - PBS phosphate-buffered saline - PtdCho phosphatidylcholine - SUV small unilamellar vesicle - Tris tris(hydroxymethyl) amino methane     

Novel mannose-binding rice lectin composed of some isolectins and its relation to a stress-inducible salT gene

The novel mannose-binding rice lectin (MRL) purified by Sephadex G-50 or maltamyl Sepharose 4B affinity chromatography was not homogeneous, but the components were separated clearly by two dimensional polyacrylamide gel electrophoresis (1st; isoelectric focusing with Immobiline, 2nd; SDS-PAGE). The major spots were located at pI 4.85 and 4.74, and minor spots at pI 4.66, 4.56, and 4.44; all spots were distributed at about MW 45,000. Other faint spots were sometimes detected just below the major spots. In the western blot analysis, all the spots reacted with the monoclonal antibodies specific to MRL, which bound to MRL and inhibited the lectin activity to agglutinate rabbit erythrocytes. The proteins of the spots at pI 4.85, 4.77, 4.66, and 4.56 had lectin activity. The major proteins at pI 4.85 and 4.77 also had the common amino acid sequence at N-terminus, TLVKIGPWGGNGGSAQDISV, which is almost identical to salt and drought stress-inducible salT gene products in rice plants. High homology was also conserved in both the cDNA and the genomic clones encoding the MRL component at pI 4.85, which were selected with MRL-specific antibodies and an oligonucleotide designed from the partial amino acid sequence. All results suggest that MRL is composed of several isolectins, if not, related proteins having a common epitope and may belong to a family of stress-inducible proteins.     

Characterization of HU-like protein from Bifidobacterium longum

A HU-like protein (HBl) of Bifidobacterium longum was purified and characterized. HBl is heat-stable and acid-resistant, and has a molecular weight of about 9.1 kDa as estimated by its mobility on electrophoresis. HBl is intermediate in basicity (pI 9.8) between the HU-1 and HU-2 proteins of Escherichia coli, and is dissociated from a calf thymus DNA-cellulose column at 300-400 mM NaCl. Its amino acid composition shows many similarities with that of E coli HU. The NH2-terminal amino acid sequence of HBl also shows significant similarities to the consensus sequence deduced from the sequences of eleven HU-like proteins from prokaryotic sources. Chemical crosslinking analysis indicated that the HBl protein predominantly forms a homotypic dimer.     

Analysis of the adenovirus type 5 proteome by liquid chromatography and tandem mass spectrometry methods

We compared detection sensitivity and protein sequence coverage of the adenovirus type 5 proteome achievable by liquid chromatography and tandem mass spectroscopy (LC/MS/MS) using three sample preparation and clean up methods. Tryptic digestion was performed on either purified viral proteins or whole virus, and followed by shotgun sequencing using tandem mass spectrometry for peptide identification. We used a recombinant adenovirus type 5 as a test system. The methods included separation of adenoviral proteins by reversed-phase high-performance liquid chromatography followed by tryptic digestion and analysis by LC/MS/MS. Alternatively, the purified whole virus was digested with trypsin and the peptides separated either by one-dimensional (reversed-phase) or by two-dimensional (cation exchange and reversed-phase) chromatography and analyzed by tandem mass spectrometry. A total of 11 protein species were identified from 154 peptides. All of the major viral proteins were found. In addition, two minor proteins, the 23 kDa viral protease and the late L1 protein, were identified for the first time by chromatography based assays. The 23 kDa viral protease, present at only 10 copies per virus, and representing 0.2% of the protein content of the virus, was detected by the 2D LC/MS/MS analysis of the whole virus digest from a sample containing only 70 fmols of the protein. This demonstrates the high sensitivity and selectivity of the method. The 2D LC/MS/MS analysis of the whole virus digest was also able to detect all viral proteins with copy numbers at or above 10/virus particle, with broad coverage of the amino acid sequences. Coverage ranged from 2 to 54%, a majority between 20 and 35%, suggesting the possibility of using this analysis to assess the purity of the virus preparations. This broad coverage may also provide a useful approach to identify posttranslational modifications on the structural proteins of the adenovirus.     

Adenovirus type 2 coded single-stranded DNA binding protein: in vivo phosphorylation and modification.

The adenovirus type 2-coded single-stranded DNA binding protein (DBP) was shown to be a phosphoprotein and to exist in at least two forms that differ in mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After a 30-min pulse with [35S]methionine or 32PO4, 35S- or 32P-labeled DBP had a nominal molecular weight of 74,000 whereas after a 30-min label followed by a 20-h chase, 35S- and 32P-labeled DBP had a nominal molecular weight of 77,000. Both large and small forms of 35S- and 32P-labeled DBP bound to single-stranded DNA-cellulose columns and were eluted by 0.4 to 0.6 M NaCl; both forms also were immunoprecipitated by antiserum against adenovirus type 1-simian virus 40-induced tumor cells (this antiserum contains antibodies against DBP) and by monospecific antiserum against 95 to 99% purified DBP. With highly purified 32P-DBP labeled 7 to 10 h postinfection, it was shown that the 32P radioactivity was firmly associated with protein material (i.e., not contaminating nucleic acids or phospholipids) and had properties expected of a phosphoester of an amino acid; paper electrophoresis of acid hydrolysates of this preparation identified phosphoserine but not phosphothreonine. Phosphoserine but not phosphothreonine was also identified in acid hydrolysates of another preparation of 32P-DBP labeled for 30 min, chased for 20 h, and then immunoprecipitated by adenovirus type 1-simian virus 40 antiserum.     

Characterization of Potent Fibrinolytic Enzymes in Earthworm,Lumbricus rubellus

Stable and potent fibrinolytic enzymes (six homogeneous proteins) were purified to homogeneity from extracts of the lyophilized powder of an earthworm, Lumbricus rubellus. The molecular weight of each enzyme estimated by SDS–polyacrylamide gel electrophoresis was different from those by gel filtration chromatography in the six purified proteins. The exact molecular weight of each enzyme (F-III-2, F-III-1, F-II, F-I-2, F-I-l, and F-I-0) measured by ionspray MS analysis was 29, 662, 29, 667, 24, 664, 24, 220, 24, 196, and 23,013, respectively. The isoelectric point (pI) of each enzyme was 3.40, 3.60, 4.20, 4.00, 4.30, and 4.85, respectively. The enzymes were single polypeptide chains. They had a very strong fibrinolytic activity and the maximum reactivity for chromogenic substrates from pH 9-11. The enzymes, acidic proteins that had abundant asparagine and aspartic acid, and low lysine in their amino acid composition, did not contain component sugars. The enzymes were stable at from pH 1-11 and up to 60°C. Studies on substrate specificity and inhibition indicated that these enzymes were alkaline trypsin-like serine proteases. N-Terminal amino acid sequences of the enzymes had local similarities to those of trypsin-like enzymes such as elastase and coagulation factor IX. From the results of amino acid sequence, amino acid composition analyses and immunological analyses, it was suggested that these six enzyme proteins were derived as isozyme(s) from at least four different genes.     

Purification of the Atlantic salmon hepatic 21 kDa protein and classification as a high mobility group chromatin protein.

The 21 kDa protein of liver from Atlantic salmon (Salmo salar) has been purified. Hepatic nuclei were extracted with 0.75 M HClO4. The extracted proteins were fractionated using reversed phase high performance liquid chromatography. The purity of the protein was analysed by isoelectric focusing in the first, and SDS-polyacrylamide gel electrophoresis in the 2nd dimension. Isoelectric focusing separated the protein into 5 spots. In gel trypsin digestion after isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis resulted in identical migration of the tryptic peptides. The amino acid composition of the 21 kDa protein was similar to that of high mobility group (HMG) proteins C and D from rainbow trout (Oncorhynchus mykiss). The N-terminal sequence of the amino acids 1-19 revealed a conserved region characteristic for HMG 14/17 proteins of mammals and avians, and their equivalents in rainbow trout. Considering the electrophoretic mobility, amino acid composition and N-terminal amino acid sequence it is concluded that the 21 kDa protein of Atlantic salmon is a member of the HMG protein family resembling the HMG D protein of rainbow trout.     

Vaccinia virus gene H5R encodes a protein that is phosphorylated by the multisubstrate vaccinia virus B1R protein kinase.

Vaccinia virus gene B1R encodes a protein kinase, the previously identified substrates of which include the proteins S2 and Sa of 40S ribosomal subunits. This work characterizes another substrate of the B1R kinase: a 36-kDa protein induced at the early stage of infection. Partially purified 36-kDa protein, eluted from a single-stranded DNA-cellulose column by 0.5 M NaCl, was separated by two-dimensional gel electrophoresis. Phosphorylation in vitro yielded multiple forms of the 36-kDa protein with approximate isoelectric points (pI) of 5.5, 5.7, 5.9, and 6.3, in addition to the apparently unphosphorylated form with a pI of approximately 6.8. The tryptic peptides derived from 36-kDa proteins with pI values of 5.7, 5.9, and 6.3 yielded almost identical high-pressure liquid chromatography profiles, strongly suggesting that the 36-kDa protein was modified by the phosphorylation of at least four sites, which were characterized as threonine residues. The amino acid sequence of several tryptic peptides derived from the 36-kDa protein showed that the 36-kDa protein was encoded by gene H5R of vaccinia virus. Consistent with this, the B1R kinase--either expressed in Escherichia coli or highly purified from HeLa cells--phosphorylated a recombinant trpE-H5R fusion protein in vitro. Fingerprints of the trpE-H5R and 36-kDa proteins phosphorylated by recombinant B1R kinase revealed common sites of phosphorylation, although some tryptic peptides were specific to either protein. Comparison was made of fingerprints of tryptic phosphopeptides derived from 36-kDa single-stranded DNA-binding protein labelled in vivo or in vitro. A common subset of peptides was observed, suggesting that some sites on H5R protein are phosphorylated by the B1R kinase in infected cells. These results suggest that some of the multiple threonine sites in the H5R protein are phosphorylated in vivo by the B1R protein kinase.     

Purification and properties of an 18-kilodalton, 1,25-dihydroxyvitamin D3 modulated protein from embryonic chick intestine

An 18,000-dalton protein (pI = 5.1) shown previously to be modulated by 1,25-dihydroxyvitamin D3 was purified to allow its further characterization. This protein from embryonic chick intestine was shown to comigrate during two-dimensional electrophoresis with an abundant protein from the intestine of 4-week-old chickens. The protein was purified from 4-week chick intestine and analyzed for amino acid composition, and 28 amino acids of its N-terminal sequence were determined. The N-terminal amino acid sequence had significant homology to cellular retinol binding protein II, an intestinal protein that has been recently sequenced. The purified 18-kilodalton protein was shown to bind retinol by fluorescence spectrophotometry. This 18-kilodalton protein is dramatically changed by 1,25-dihydroxyvitamin D3 in the chick embryonic organ culture system. Therefore, further study of it may lead to a better understanding of vitamin A and D interaction and how 1,25-dihydroxyvitamin D3 acts through proteins to stimulate intestinal calcium and phosphate transport.     

A study of the interaction of human spermatozoa membrane with ATP and cyclic-AMP

The three coat proteins of foot-and-mouth disease virus type O can be separated in milligram amounts using a SE-cellulose ionic exchanger, both from each other and viral RNA not quantitatively precipitated by acetic acid. The amino acid composition of the separated proteins has been determined and a statistical method has been used to calculate, for each, the most probable number of amino acids and molecular weight. The N-terminal amino acids of the three proteins are glycine, asparagine and threonine.     

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.     

Isolation and comparative biochemical properties of the major internal polypeptides of equine infectious anemia virus.

We describe procedures for the large-scale production of equine infectious anemia virus (EIAV) and for the isolation of the four major non-glycosylated virion proteins, designated p26, p15, p11, and p9. Comparisons of the purified proteins by peptide mapping procedures and by enzyme-linked immunosorbent assays demonstrated the unrelatedness of the four proteins. The characteristic properties of each purified protein were examined by determining isoelectric points and amino acid compositions. We found that EIAV p26 and p9 focus at pI values of 6.2 and 5.0, respectively, and that these proteins contain no unusual amino acids. In contrast, EIAV p15 reproducibly displayed a heterogeneous isoelectric focusing pattern, with major pI values ranging from 5.7 to 8.3. This charge variation evidently correlated with different levels of phosphorylated serine or threonine or both, which could be detected by an amino acid analysis of purified p15. EIAV p11 apparently focused at a pI of greater than 10, reflecting its high content of basic amino acids. Moreover, localization experiments indicated that all four nonglycosylated proteins constitute the internal components of the virus, with all of the virion p11 closely associated with the viral RNA genome. Thus, our results demonstrated that EIAV, a lentivirus, contains structural polypeptides which are analogous to the structural polypeptides described previously in prototype C oncoviruses.     

Die Proteinstruktur des RNS-Bakteriophagen fr

Summary The coat protein of the RNA containing bacteriophage f_r has been hydrolyzed and its amino acid composition determined (Table 1). Furthermore, the protein was split with trypsin and the tryptic peptides were separated by column chromatography on Dowex 1 (Figure 1) and purified by paper chromatography and electrophoresis.The amino acid composition of all but one tryptic peptide are given in Table 2. The large peptide T₁₃ which is much more difficult to purify than all other peptides, was isolated by several methods. Its amino acid composition is shown in Table 3. All tryptic peptides are compiled in Table 4.Amino acid sequences have been fully or partially determined for 9 tryptic peptides (Table 5) and the others are presently being investigated.These findings are compared with the results from other RNA phages, especially f₂. It is concluded from the available data that the relationship between the coat proteins of the RNA phages is similar to that between the various naturally occurring strains of tobacco mosaic virus whose amino acid sequences are known.

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Herrn Prof.G. Melchers zum 60. Geburtstag gewidmet.     

High performance liquid chromatography and characterization of structural proteins of the influenza virus

Constituent proteins of human influenza virus A have been separated by reverse-phase HPLC on Polysil ODS-500. Their homogeneity is confirmed by the data of amino acid composition, Edman analysis and gel electrophoresis.     

Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that contain epsilon-N-acetyllysine.

Recombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, epsilon-N-acetyllysine. This amino acid was initially discovered in place of the normal lysine144 in a modified reversed-phase HPLC (RP-HPLC) species of rpST. Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP-HPLC purified protein were used to identify this altered residue as epsilon-N-acetyllysine. Ion-exchange chromatography was utilized to prepare low isoelectric point (pI) forms of rpST and rbST, which are enriched in epsilon-N-acetyllysine. Electrospray mass spectrometry demonstrated that the majority of the protein in these low pI fractions contained species 42 Da larger than normal. Immobilized pH gradient electrophoresis (IPG) of the ion-exchange purified low pI proteins was used to isolate several monoacetylated species of rpST and rbST. The location of the acetylated lysine in each IPG-purified protein was determined by tryptic peptide mapping and amino acid sequencing of the altered tryptic peptides. Amino acid analyses of enzymatic digests of rpST and rbST were also used to confirm the presence of epsilon-N-acetyllysine in these recombinant proteins. These data demonstrate that a significant portion of rpST and rbST produced in E. coli contain this unusual amino acid.     

Isolation and Characterization of Two Protein Isoforms with Antiviral Activity from Chenopodium album L Leaves

Two antiviral proteins (AVPs) named CAP-I and CAP-II isolated and purified from the leaves of Chenopodium album cv Pusa Bathua 1 were found to inhibit tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) infection on their respective host plants. The molecular weight of both the AVPs was found to be 24 kD. They were devoid of carbohydrate moiety and were highly basic with pI ～10.2. However, they differed with respect to amino acid composition and N-terminal sequence. They also differed with respect to IC₅₀ values, and CAP-I was found to be 2.5 fold more effective than CAP-II in inhibiting viral infection.     

赤子爱胜蚓（Eisenia foetida）中抗肿瘤与纤溶酶原激酶活性蛋白质的分离与鉴定

采用SephadexG 75和HiPrep 1 6 / 6 0DEAE离子交换等方法从赤子爱胜蚓 (Eiseniafoetida)中提取到一组活性蛋白质成分 ,双向电泳分析证明它们为一组pI在 3 .0～ 4 .0之间的酸性蛋白质 ;利用体外K5 6 2、HeLa、SY5Y等肿瘤细胞抑制实验和纤维蛋白平板实验 ,跟踪测定活性 ,证明乙醇沉淀组分D2 (8)是既具肿瘤抑制 ,又具有激酶活性的蛋白质成分。同时应用非变性电泳对乙醇沉淀组分进行分离 ,并利用电洗脱、凝胶原位酶解和ESI MS等蛋白质组学方法 ,鉴定了其中 6种蛋白质的分子量、氨基酸组成、N末端序列和肽质量指纹图信息 ,其中条带 9与D2 (8)组分为同一种蛋白质 ;研究证明蚯蚓中含有既具抗肿瘤活性又具有激酶活性的蛋白质成分。采用的方法可适用于活性蛋白质成分的整体分离与鉴定