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Article: Peptide analysis of collagen produced from cDNA by transcription and translation in vitro.

TitlePeptide analysis of collagen produced from cDNA by transcription and translation in vitro.
Authors
Issue Date1987
PublisherPortland Press Ltd. The Journal's web site is located at http://www.biochemj.org
Citation
Biochemical Journal, 1987, v. 245 n. 2, p. 393-398 How to Cite?
AbstractWhen collagen CNBr-cleavage peptides are analysed by two-dimensional gel electrophoresis each peptide is resolved into a reproducible set of charged forms. To test whether this peptide heterogeneity resulted from polymorphic mRNA, collagen was produced by transcription and translation in vitro of a collagen cDNA clone, and the peptides were mapped by two-dimensional gel electrophoresis. A cDNA construct was produced by ligation of the 5' end of the rat phenylalanine hydroxylase cDNA [Dahl & Mercer (1986) J. Biol. Chem. 261, 4148-4153], containing the translation-initiation codon, to a human alpha 1(I) cDNA [Chu, Myers, Bernard, Ding & Ramirez (1982) Nucleic Acids Res. 10, 5925-5934] coding for a large portion of helical region including the complete CB7 and CB3 CNBr-cleavage peptides. This cDNA construct was ligated into the transcription vector pSP65, and cell-free translation of the mRNA transcribed from the pSP65 plasmid was performed with a rabbit reticulocyte lysate system. After CNBr cleavage of the hybrid protein translation products, the collagen CB7 and CB3 peptides were resolved by two-dimensional electrophoresis into the same multiple charged forms whether the mRNA was produced from the cDNA construct or was extracted from normal fibroblast cultures. This result demonstrated that the multiple peptide spots were not due to polymorphic mRNA species. The heterogeneity must result from some uncharacterized specific post-translational modification or chemical alterations during sample preparation. This method of expression and analysis of proteins from cDNA clones should be of considerable use in the identification and characterization of clones that code for mutant proteins.
Persistent Identifierhttp://hdl.handle.net/10722/147327
ISSN
2015 Impact Factor: 3.562
2015 SCImago Journal Rankings: 2.582
ISI Accession Number ID

 

DC FieldValueLanguage
dc.contributor.authorBateman, JFen_US
dc.contributor.authorLamande, Sen_US
dc.contributor.authorChan, Den_US
dc.contributor.authorCole, WGen_US
dc.date.accessioned2012-05-29T06:02:56Z-
dc.date.available2012-05-29T06:02:56Z-
dc.date.issued1987en_US
dc.identifier.citationBiochemical Journal, 1987, v. 245 n. 2, p. 393-398en_US
dc.identifier.issn0264-6021en_US
dc.identifier.urihttp://hdl.handle.net/10722/147327-
dc.description.abstractWhen collagen CNBr-cleavage peptides are analysed by two-dimensional gel electrophoresis each peptide is resolved into a reproducible set of charged forms. To test whether this peptide heterogeneity resulted from polymorphic mRNA, collagen was produced by transcription and translation in vitro of a collagen cDNA clone, and the peptides were mapped by two-dimensional gel electrophoresis. A cDNA construct was produced by ligation of the 5' end of the rat phenylalanine hydroxylase cDNA [Dahl & Mercer (1986) J. Biol. Chem. 261, 4148-4153], containing the translation-initiation codon, to a human alpha 1(I) cDNA [Chu, Myers, Bernard, Ding & Ramirez (1982) Nucleic Acids Res. 10, 5925-5934] coding for a large portion of helical region including the complete CB7 and CB3 CNBr-cleavage peptides. This cDNA construct was ligated into the transcription vector pSP65, and cell-free translation of the mRNA transcribed from the pSP65 plasmid was performed with a rabbit reticulocyte lysate system. After CNBr cleavage of the hybrid protein translation products, the collagen CB7 and CB3 peptides were resolved by two-dimensional electrophoresis into the same multiple charged forms whether the mRNA was produced from the cDNA construct or was extracted from normal fibroblast cultures. This result demonstrated that the multiple peptide spots were not due to polymorphic mRNA species. The heterogeneity must result from some uncharacterized specific post-translational modification or chemical alterations during sample preparation. This method of expression and analysis of proteins from cDNA clones should be of considerable use in the identification and characterization of clones that code for mutant proteins.en_US
dc.languageengen_US
dc.publisherPortland Press Ltd. The Journal's web site is located at http://www.biochemj.orgen_US
dc.relation.ispartofBiochemical Journalen_US
dc.subject.meshCollagen - Geneticsen_US
dc.subject.meshDna - Geneticsen_US
dc.subject.meshElectrophoresis, Polyacrylamide Gelen_US
dc.subject.meshPeptide Fragments - Analysisen_US
dc.subject.meshProtein Biosynthesisen_US
dc.subject.meshRna, Messenger - Geneticsen_US
dc.subject.meshTranscription, Geneticen_US
dc.titlePeptide analysis of collagen produced from cDNA by transcription and translation in vitro.en_US
dc.typeArticleen_US
dc.identifier.emailChan, D:chand@hkucc.hku.hken_US
dc.identifier.authorityChan, D=rp00540en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.pmid3663165en_US
dc.identifier.scopuseid_2-s2.0-0023655452en_US
dc.identifier.volume245en_US
dc.identifier.issue2en_US
dc.identifier.spage393en_US
dc.identifier.epage398en_US
dc.identifier.isiWOS:A1987J184200012-
dc.publisher.placeUnited Kingdomen_US
dc.identifier.scopusauthoridBateman, JF=16135557700en_US
dc.identifier.scopusauthoridLamande, S=7004500719en_US
dc.identifier.scopusauthoridChan, D=7402216545en_US
dc.identifier.scopusauthoridCole, WG=7201518727en_US

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