Genetic engineering and overexpression of ribosomal L12 protein genes from three different archaebacteria in E coli
Genes coding for ribosomal protein L12 from Methanococcus vannielii (Mva), Halobacterium halobium (Hha) and Sulfolobus solfataricus (Sso) have been subcloned in the polylinker region of pUC19. An efficient Shine-Dalgarno sequence has been attached to the 5′ end of the genes, and two ochre stop codon...
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Published in | Biochimie Vol. 73; no. 6; pp. 647 - 655 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
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Elsevier Masson SAS
01.06.1991
Elsevier |
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Abstract | Genes coding for ribosomal protein L12 from
Methanococcus vannielii (Mva),
Halobacterium halobium (Hha) and
Sulfolobus solfataricus (Sso) have been subcloned in the polylinker region of pUC19. An efficient Shine-Dalgarno sequence has been attached to the 5′ end of the genes, and two ochre stop codons have been created at their 3′ ends, where necessary. In addition, mutants of the MvaL12 and HhaL12 genes were constructed, which coded for a cysteine residue at the C-terminus of the protein. The constructs were transferred together with the pUC19 polylinker as gene cartridges into different expression vectors. These constructed plasmids were transformed in the appropriate
E coli hosts and tested for expression. Two systems were found to work efficiently for overexpression, namely the pKK223-3 vector eaturing a
tac promoter, and the pT7-5 vector featuring a T7-promoter. The overexpressed proteins were purified to homogeneity; their purity was investigated by one and two-dimensional gel systems, amino acid analysis and N-terminal protein sequencing for 10 steps or more. The amount of protein purified from
E coli test cultures bearing the expression plasmids was always more than 2.5 mg/l of medium used. |
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AbstractList | Genes coding for ribosomal protein L12 from Methanococcus vannielii (Mva), Halobacterium halobium (Hha) and Sulfolobus solfataricus (Sso) have been subcloned in the polylinker region of pUC19. An efficient Shine-Dalgarno sequence has been attached to the 5' end of the genes, and two ochre stop codons have been created at their 3' ends, where necessary. In addition, mutants of the MvaL12 and HhaL12 genes were constructed, which coded for a cysteine residue at the C-terminus of the protein. The constructs were transferred together with the pUC19 polylinker as gene cartridges into different expression vectors. These constructed plasmids were transformed in the appropriate E coli hosts and tested for expression. Two systems were found to work efficiently for overexpression, namely the pKK223-3 vector featuring a tac promoter, and the pT7-5 vector featuring a T7-promoter. The over-expressed proteins were purified to homogeneity; their purity was investigated by one and two-dimensional gel systems, amino acid analysis and N-terminal protein sequencing for 10 steps or more. The amount of protein purified from E coli test cultures bearing the expression plasmids was always more than 2.5 mg/l of medium used. Genes coding for ribosomal protein L12 from Methanococcus vannielii (Mva), Halobacterium halobium (Hha) and Sulfolobus solfataricus (Sso) have been subcloned in the polylinker region of pUC19. An efficient Shine-Dalgarno sequence has been attached to the 5′ end of the genes, and two ochre stop codons have been created at their 3′ ends, where necessary. In addition, mutants of the MvaL12 and HhaL12 genes were constructed, which coded for a cysteine residue at the C-terminus of the protein. The constructs were transferred together with the pUC19 polylinker as gene cartridges into different expression vectors. These constructed plasmids were transformed in the appropriate E coli hosts and tested for expression. Two systems were found to work efficiently for overexpression, namely the pKK223-3 vector eaturing a tac promoter, and the pT7-5 vector featuring a T7-promoter. The overexpressed proteins were purified to homogeneity; their purity was investigated by one and two-dimensional gel systems, amino acid analysis and N-terminal protein sequencing for 10 steps or more. The amount of protein purified from E coli test cultures bearing the expression plasmids was always more than 2.5 mg/l of medium used. |
Author | Hannemann, F. Köpke, A.K.E. Boeckh, T. |
Author_xml | – sequence: 1 givenname: A.K.E. surname: Köpke fullname: Köpke, A.K.E. organization: Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Ihnestrasse 73, D-1000 Berlin 33, Germany – sequence: 2 givenname: F. surname: Hannemann fullname: Hannemann, F. organization: Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Ihnestrasse 73, D-1000 Berlin 33, Germany – sequence: 3 givenname: T. surname: Boeckh fullname: Boeckh, T. organization: Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Ihnestrasse 73, D-1000 Berlin 33, Germany |
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Keywords | ribosomal proteins protein purification gene modifications overexpression DTE DMF Purification Archaeobacteria Escherichia coli HPLC chromatography Gene expression Halobacterium halobium Ribosomal protein Biological activity Ion exchange chromatography Gene Bacteria Two dimensional electrophoresis Mutation Halobacteriaceae Enterobacteriaceae |
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Snippet | Genes coding for ribosomal protein L12 from
Methanococcus vannielii (Mva),
Halobacterium halobium (Hha) and
Sulfolobus solfataricus (Sso) have been subcloned... Genes coding for ribosomal protein L12 from Methanococcus vannielii (Mva), Halobacterium halobium (Hha) and Sulfolobus solfataricus (Sso) have been subcloned... |
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SubjectTerms | Amino Acids - analysis Archaea - genetics Base Sequence Biological and medical sciences Biotechnology Chromatography, Ion Exchange Escherichia coli Escherichia coli - genetics Fundamental and applied biological sciences. Psychology Gene Expression Regulation, Bacterial gene modifications Genetic Engineering Genetic technics Genetic Vectors Methods. Procedures. Technologies Miscellaneous Molecular Sequence Data Mutagenesis, Site-Directed Oligonucleotides - chemical synthesis overexpression protein purification ribosomal proteins Ribosomal Proteins - biosynthesis Ribosomal Proteins - genetics Ribosomal Proteins - isolation & purification Synthetic digonucleotides and genes. Sequencing |
Title | Genetic engineering and overexpression of ribosomal L12 protein genes from three different archaebacteria in E coli |
URI | https://dx.doi.org/10.1016/0300-9084(91)90044-2 https://www.ncbi.nlm.nih.gov/pubmed/1764512 https://search.proquest.com/docview/16225665 |
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