An immunoaffinity purification procedure for sv40 large t antigen
VirologyPublished 1 July 1985
Viesturs Simanis, David P. Lane
Citations289
SJR quartileQ2
SJR score0.74
SNIP0.60
Generate an AI Snapshot to get a quick, structured summary of this paper.
Study Snapshot
ObjectiveStudy objective
MethodsResearch methodology
PopulationPopulation studied
Sample sizeSample sizes
OutcomesStudy outcomes here
ResultsStudy results comes here
LimitationsResearch study limitations comes here
A concise AI-generated summary of the paper will appear here once you click Generate AI Snapshot.
TL;DR
A rapid purification procedure has been developed which combines the use of an adenovirus-SV40 hybrid virus which overproduces large T antigen, and immunoaffinity chromatography on an anti-large T monoclonal antibody coupled to protein A Sepharose, allowing the isolation of milligram amounts of large T in excellent yield.
Abstract
A rapid purification procedure for SV40 large T antigen has been developed which combines the use of an adenovirus-SV40 hybrid virus which overproduces large T antigen, and immunoaffinity chromatography on an anti-large T monoclonal antibody coupled to protein A Sepharose. The protein exhibits the p53-binding, ATPase, and sequence-specific DNA-binding activities of T antigen. The purification procedure can be completed in 1 day and allows the isolation of milligram amounts of large T in excellent yield. The pure protein is extremely antigenic and is tolerant of iodination to high specific activity, permitting the development of a competition radioimmunoassay for large T that reliably detects nanogram amounts of the protein.
Keywords
MedicineBiochemistry, Genetics and Molecular Biology
NatureCleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4
251,640 Citations1970Ulrich K. Laemmli
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products.
Proceedings of the National Academy of SciencesElectrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
53,843 Citations1979Harry Towbin, T. Staehelin +1 more
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets that results in quantitative transfer of ribosomal proteins from gels containing urea.
Analytical BiochemistryA simplification of the protein assay method of Lowry et al. which is more generally applicable
8,545 Citations1977Gary L. Peterson
A simple method based on a linear log-log protein standard curve is presented to permit rapid and totally objective protein analysis using small programmable calculators.
GeneA new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments
2,941 Citations1982Joachim Messing, Jeffrey Vieira
Two new M13 vectors, M13MP8 and M13mp9, have been constructed that permit the cloning of the same restriction fragment in both possible orientations, allowing the use of only one of the two DNA strands as a template for M13 shotgun sequencing.
NatureT antigen is bound to a host protein in SY40-transformed cells
2,413 Citations1979David P. Lane, L.V. Crawford
It is reported here that the T antigen in a line of SV40-transformed mouse cells forms an oligomeric complex with a specific cell coded protein.
Nucleic Acids ResearchDNAase footprinting a simple method for the detection of protein-DNA binding specificity
2,120 Citations1978David J. Galas, Albert Schmitz
Journal of VirologyMonoclonal antibodies specific for simian virus 40 tumor antigens
1,105 Citations1981Ed Harlow, L.V. Crawford +2 more
Thirty hybridomas that secrete immunoglobulins against the simian virus 40 tumor antigens were isolated and cloned and large-T antigen was found to have at least one determinant that it shared with small-t antigen and to have a minimum of six unique determinants.
Die NaturwissenschaftenDie Molekularbiologie der Tumorviren
944 Citations1977Gerhard Sauer
All classes of vertebrates harbor tumor viruses that are capable of inducing either tumors or leukemias, and these viruses have already been assigned to specific regions of their DNA.
Journal of Biological ChemistryA one-step purification of membrane proteins using a high efficiency immunomatrix.
770 Citations1982C. Schneider, Roland Newman +3 more
A method is described by which an immunoaffinity matrix was constructed by binding antibody directly or indirectly to protein A-Sepharose 4B followed by cross-linking of the complex with dimethyl pimelimidate, which allows optimal spatial orientation of antibodies and, thus, maximum antigen binding efficiency.
Journal of VirologySimian Virus 40 Deoxyribonucleic Acid Synthesis: the Viral Replicon
603 Citations1972P Tegtmeyer
Temperature-shift experiments and analysis of SV40 viralDNA replication by gel electrophoresis have provided strong evidence that the ts gene product of the three mutants is directly required to initiate each new round of viral DNA replication but is not required to complete a cycle which has already begun.
ElectrophoresisUltrasensitive silver‐based color staining of polypeptides in polyacrylamide gels
596 Citations1981David W. Sammons, Lonnie D. Adams +1 more
The sensitivity of the silver stain process is superior to Coomassie Blue and is comparable to autoradiography after in corporation of conventional levels of 35S‐ methionine.
Journal of VirologyRegulation of tumor antigen synthesis by simain virus 40 gene A
432 Citations1975P Tegtmeyer, Martin A. Schwartz +2 more
The presence of the same virus-induced protein in infected cells from different species and the altered behavior of that protein in mutant infection strongly suggest that the protein is virus coded.
CellActivation of the SV40 late promoter: Direct effects of T antigen in the absence of viral DNA replication
292 Citations1984Janis M. Keller, James C. Alwine
It is demonstrated that T antigen can substantially stimulate late promoter activity directly and independent of viral DNA replication.
CellActivation of mouse genes in transformed cells
253 Citations1983Michael R. Scott, K. H. Westphal +1 more
Proceedings of the National Academy of SciencesAutoregulation of simian virus 40 gene A by T antigen.
247 Citations1976Steven I. Reed, George R. Stark +1 more
It is suggested that the A protein regulates its own synthesis by negative feedback control of gene A transcription, which is translated into A protein (T antigen) during lytic infection by simian virus.
CellT antigen repression of SV40 early transcription from two promoters
244 Citations1981Ulla Hansen, Daniel G. Tenen +2 more
D2T, a T antigen analog, specifically represses transcription in vitro from both initiation sites, but at different concentrations, whereas D2T to site I suppresses initiation from the site downstream of the TATA box.
Journal of Molecular BiologyBinding of a simian virus 40 T antigen-related protein to DNA
232 Citations1981Ronald D.G. McKay
Another region of the SV40 genome showed preferential binding to D2T, however, the decay rate of this site in the EcoRII/D fragment was rapid and, under these conditions, could not be distinguished from non-specific binding.
Journal of VirologyTopography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication
226 Citations1983A L DeLucia, B A Lewton +2 more
Investigation of the DNA binding properties of the simian virus 40 (SV40) A protein (large T antigen) and the hybrid adenovirus-SV 40 D2 protein revealed that both viral proteins protect similar regions of SV40 DNA from digestion by DNase I or methylation by dimethyl sulfate.
NatureSV40 large T shares an antigenic determinant with a cellular protein of molecular weight 68,000
213 Citations1980David P. Lane, Warren K. Hoeffler
To clarify the action of T proteins in transformation, several monoclonal antibodies directed against different regions of the T molecule are prepared and one of these antibodies recognizes an antigenic determinant on T that is also present on a host cell protein of molecular weight 68,000.
Proceedings of the National Academy of SciencesBiological activity of purified simian virus 40 T antigen proteins.
197 Citations1978Robert Tjian, G H Fey +1 more
Proteins related to simian virus 40 (SV40) T antigen and proteins that share sequences with it retain the ability to carry out at least two functions associated with the product of the A gene of SV40.
Journal of VirologyModification of simian virus 40 protein A
167 Citations1977P Tegtmeyer, Kathleen Rundell +1 more
A study of extraction conditions shows that the smaller proteins are easily generated during extraction and provides an explanation for the appearance of these proteins in some cells after extraction under unfavorable conditions.
Journal of VirologyCharacterization of the Autoregulation of Simian Virus 40 Gene <i>A</i>
167 Citations1977James C. Alwine, Steven I. Reed +1 more
The observations are consistent with a simple model in which the binding of A protein at the origin of replication blocks either binding of RNA polymerase to the early promoter or its progress through the early gene(s).
Journal of Biological ChemistryUse of monoclonal antibodies as probes of simian virus 40 T antigen ATPase activity.
143 Citations1981R Clark, David P. Lane +1 more
The results indicate that monoclonal antibodies can be used as probes of enzyme structure and function and suggest that the ATPase function of T antigen maps on the SV40 A gene between 0.37 and 0.29 map unit.
CellSV40 T antigen binding site mutations that affect autoregulation
136 Citations1983Donald C. Rio, Robert Tjian
Results suggest a functional role for T antigen binding site II in the repression of early SV40 transcription and indicate that binding of T antigen to both sites I and II plays a role in autoregulation.
Journal of VirologyDetection and characterization of multiple forms of simian virus 40 large T antigen
133 Citations1981Ellen Fanning, Paweł Nowak +1 more
Different biological functions of large T antigen may be carried out by different subclasses of this protein, as reported recently in simian virus 40-transformed and -infected cells separated by zone velocity sedimentation in sucrose density gradients.
CellEssential contact residues within SV40 large T antigen binding sites I and II identified by alkylation-interference
126 Citations1984Keith Jones, Robert Tjian
Three-dimensional projection reveals that the guanine contacts within each binding site are localized so that the specific binding interactions are accessible from only one face of the DNA helix.
CellDifferent structural systems of the nucleus are targets for SV40 large T antigen
124 Citations1983Matthias Staufenbiel, Wolfgang Deppert
Different solubility properties, differences in the amount of the cellular phosphoprotein p53 coprecipitating with large T, and different stabilities in its association with the nuclear structural systems indicate that distinct subclasses of large T were isolated from their in vivo location in SV40-transformed cells.
Proceedings of the National Academy of SciencesRegulation of human ribosomal RNA transcription.
115 Citations1983R. Marc Learned, Stephen T. Smale +2 more
Analysis of deletion mutants spanning the start site of transcription at nucleotide +1 indicates that the control region affecting initiation of human rRNA synthesis is contained within sequences from nucleotides -158 to +18.
CellInitiation points for DNA replication in nontransformed and simian virus 40-transformed Chinese hamster lung cells
112 Citations1977Robert G. Martin, Ariella Oppenheim
Randomly growing Chinese hamster lung cells were pulse-labeled with 3H-thymidine, and the replicating forks of individual DNA fibers were visualized by autoradiography to compare wild-type SV40-transformed cells with nontransformed cells.
CellModification of SV40 T antigen by poly ADP-ribosylation
106 Citations1981Neil D. Goldman, Myles Brown +1 more
A novel modification of a population of SV40 T antigen molecules by poly ADP-ribosylation is demonstrated, suggesting a similar role in the regulation of certain biological functions under the control of this protein.
Proceedings of the National Academy of SciencesLarge and small tumor antigens from simian virus 40 have identical amino termini mapping at 0.65 map units.
104 Citations1978E Paucha, Andrew L. Mellor +4 more
Partial amino-terminal amino acid sequences of both large and small tumor (T)antigens of simian virus 40 were determined and found to be identical, which strongly suggests that the sequence coding for the amino terminus of both proteins is located at this position.
Journal of VirologyCellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen
103 Citations1982L. Crawford, Keith N. Leppard +2 more
Several recently isolated monoclonal antibodies which reacted with simian virus 40 T antigens also reacted with proteins found in uninfected and untransformed cells, suggesting similarities of shape and perhaps function between domains of the viral T antigen and the relevant host proteins.
Advances in cancer researchThe Transformation of Cell Growth and Transmogrification of DNA Synthesis by Simian Virus 40
102 Citations1981Robert G. Martin
The hypothesis is that transformation by SV40 unlike PyV is largely explicable in terms of the ability of the 90K T-antigen to initiate DNA synthesis, which can propel the cell into rounds of replication beyond those exhibited by non-transformed cells in the same conditions.
CellSV40 large tumor antigen can regulate some cellular transcripts in a positive fashion
101 Citations1982Ted E. Schutzbank, R. Robinson +2 more
The results demonstrate that the SV40 large T antigen can regulate the steady state levels of some cellular RNA species.
Journal of VirologySimian Virus 40 Mutants with Deletions at the 3′ End of the Early Region Are Defective in Adenovirus Helper Function
90 Citations1979Charles N. Cole, L.V. Crawford +1 more
Proceedings of the National Academy of SciencesReactivation of silent rRNA genes by simian virus 40 in human-mouse hybrid cells.
88 Citations1979Kenneth J. Soprano, V.G. Dev +2 more
Mouse-human hybrid cells were used to study the ability of simian virus 40 to regulate the expression of rRNA genes in vivo, and results indicate that simianirus 40 infection can induce theexpression of otherwise silent r RNA genes.
CellExpression of SV40 T antigen under control of adenovirus promoters
87 Citations1981Carl S. Thummel, Robert Tjian +1 more
Biochemical characterization of SV40 T antigens overproduced by the hybrid viruses indicates that they are structurally indistinguishable from wild-type SV40 large T antigen and are functionally equivalent to the D2 protein.
CellTranslational control of SV40 T antigen expressed from the adenovirus late promoter
75 Citations1983Carl S. Thummel, Robert Tjian +2 more
Some form of translational regulation is present in adenovirus-infected cells that is missing from the in vitro translation reaction.
Journal of Biological ChemistryAmino acid sequence analysis of fragments generated by partial proteolysis from large simian virus 40 tumor antigen.
71 Citations1980Martin Schwyzer, Robert Weil +2 more
Large simian virus 40 tumor antigen was bound as immune complex to protein A-Sepharose and then subjected to limited proteolysis which yielded several discrete fragments, which identified the reading frame of translation beyond the intervening sequence at 0.51 map unit of the viral genome.
CellConstruction of an adenovirus-SV40 recombinant producing SV40 T antigen from an adenovirus late promoter
61 Citations1981David Solnick
An adenovirus-SV40 recombinant has been constructed in which the SV40 early region was substituted for a small portion of the adenvirus late region and all of early region 3, and the RNAs produced lack both the second and third members of the tripartite leader normally found on adenavirus late RNA, yet are translationally active, directing the synthesis of substantial amounts of SV40 T antigen.
VirologyAlternative interactions of the SV40 a protein with DNA
60 Citations1981P Tegtmeyer, Betsy Andersen +2 more
It is proposed that the alternative interactions of SV40 A protein with the 60- to 65-bp origin fragment may represent three different conformational alignments of protein with DNA rather than a simple repetition of a single kind of binding interaction.
Journal of VirologySpecific association of simian virus 40 tumor antigen with simian virus 40 chromatin
57 Citations1980Jakob Reiser, Jaime Renart +2 more
Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific.
Journal of VirologySimian virus 40 tumor antigen: isolation of the origin-specific DNA-binding domain
56 Citations1983B Morrison, Michel Kress +2 more
In vivo phosphorylation of the T antigen within this region completely abolished the ability of the 17,000-Mr peptide to bind DNA, consistent with the idea that DNA binding by theT antigen is regulated by posttranslational modifications.
Journal of VirologyAssociation of Simian Virus 40 T Antigen with Simian Virus 40 Nucleoprotein Complexes
55 Citations1979Kristine Mann, Tony Hunter
Cold Spring Harbor Symposia on Quantitative BiologyCatalytic Properties of the SV40 Large T Antigen
47 Citations1980Robert Tjian, A K Robbins +1 more
Journal of Biological ChemistrySingle strand DNA binding of simian virus 40 tumor antigen.
46 Citations1979Thomas Spillman, Donald Giacherio +1 more
These experiments showed that T antigen binds preferentially to single strand calf thymus DNA by more than an order of magnitude when compared to double strand calfThymusDNA.
Journal of VirologyAssociation of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40
38 Citations1980M. Segawa, Sumio Sugano +1 more
It is suggested that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleop protein complexes simultaneously with completion ofDNA replication.
Journal of Molecular BiologyBinding of simian virus 40 large T antigen from virus-infected monkey cells to wild-type and mutant viral replication origins
37 Citations1983Daniel G. Tenen, Thomas S. Taylor +4 more
The binding of purified simian virus 40 (SV40) large T antigen (T) from monkey cells infected with wild-type SV40 virus to viral replication origin-containing DNA fragments was studied by DNase footprinting and restriction endonuclease protection methods, and sequences in the "early" portion of site 2 are sufficient for T binding to the intact site.
Journal of VirologyConstruction and Characterization of Viable Deletion Mutants of Simian Virus 40 Lacking Sequences near the 3′ End of the Early Region
36 Citations1982M Polvino-Bodnar, Charles N. Cole
Five viable deletion mutants of simian virus 40 (SV40) were prepared and characterized and showed reduced rates of growth, lower yields of progeny virus and viral DNA, and smaller plaque size; in these properties the mutants resembled parental SV40 mutant dl862, although mutant progeny yields were usually lower than yields of dl 862, suggesting a possible interaction between the two deletions.
NatureTightly associated lipids may anchor SV40 large T antigen in plasma membrane
29 Citations1983Roland Henning, Jutta Lange-Mutschler
T antigen extracted from unlabelled SV40-transformed cells (SV80) becomes 3H-labelled after in vitro binding to the cell surface of3H-palmitate-prelabelled HeLa cells, suggesting that T antigen attached externally to living cells, may be anchored by tightly linked lipids.
VirologyPartial purification of sv40 a protein and a related cellular protein from permissive cells
27 Citations1981P Tegtmeyer, Betsy Andersen
A simple and reproducible method for the purification of SV40 A protein (T antigen) from permissive cells has been developed and two cellular proteins are also purified in smaller quantities by the same procedure.
Proceedings of the Royal Society of London. Series B, Biological sciencesThe complex between simian virus 40 T antigen and a specific host protein
25 Citations1980David P. Lane, L.V. Crawford
The small DNA tumour virus simian virus 40 (SV40) expresses only two proteins in the cells that it has transformed, large-T and small-t, and genetic evidence indicates that it is necessary both for the induction and maintenance of the transformed state.
PubMedSubclasses of simian-virus-40 large tumor antigen. Partial purification and DNA-binding properties of two subclasses of tumor antigen from productively infected cells.
22 Citations1982Arnulf Dorn, Daria Brauer +3 more
Proceedings of the National Academy of SciencesPurification of simian virus 40 tumor antigen from a line of simian virus 40-transformed human cells.
21 Citations1977Daniel G. Tenen, H S Garewal +5 more
Simian virus 40 tumor antigen can be isolated in a highly purified state from the nuclei of SV80 cells, a continuous line of simian virus40-transformed human fibroblasts, and contains a detectable amino-terminal residue.
European Journal of BiochemistrySubclasses of Simian-Virus-40 Large Tumor Antigen
13 Citations2005Arnulf Dorn, Daria Brauer +3 more
Two major subclasses of simian virus 40 tumor antigen were prepared from productively infected monkey cells and found that the affinity of both tumor antigen forms for the viral genome control region was at least 10-fold higher than their affinity for nonspecific DNA sequences.
European Journal of BiochemistryThe Simian‐Virus‐40 Large‐Tumor Antigen in Replicating Viral Chromatin
13 Citations1983H. Stahl, Michael Bauer +1 more
It is considered the possibility that the unexpectedly tight binding of T antigen to replicating DNA is related to the function that T antigen performs during viral DNA replication.