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A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
2009 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 2, e15- p.Article in journal (Refereed) Published
Abstract [en]

With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)(6)-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)(6)-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.

Place, publisher, year, edition, pages
2009. Vol. 37, no 2, e15- p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-137336DOI: 10.1093/nar/gkn992ISI: 000262963400006OAI: oai:DiVA.org:uu-137336DiVA: diva2:378161
Available from: 2010-12-15 Created: 2010-12-15 Last updated: 2012-03-08Bibliographically approved
In thesis
1. Ribosomal Stalk Protein L12: Structure, Function and Application
Open this publication in new window or tab >>Ribosomal Stalk Protein L12: Structure, Function and Application
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ribosomal stalk proteins are known to play important role in protein synthesis. The ‘stalk’, an extended structure on the large subunit of the ribosome is composed mainly of two to three dimers of L12 and one L10 protein, which forms the base of the stalk. In E. coli, four copies of L12 molecules exist as dimer of dimers forming the pentameric L8 complex together with L10. This thesis is a collection of four interlinked studies on the structure, function and application of the ribosomal stalk protein L12. In the first study, we have mapped the interaction sites of the four major translation GTPase factors (IF2, EF-Tu, EF-G & RF3) on L12 molecule using heteronuclear NMR spectroscopy. Surprisingly, all these factors produced an overlapping interaction map spanning two α-helices on the C terminal domain of L12, thereby suggesting a general nature of the interaction between L12 and the GTPase factors. L12 is known to stimulate GTPase activity of the elongation factors EF-Tu and EF-G. Here, we have clarified the role of L12 in IF2 mediated initiation of protein synthesis. Our data suggest that rapid subunit association requires a specific interaction between the L12 protein on the 50S and IF2·GTP on the 30S preinitiation complex. We have also shown that L12 is not a GAP for IF2 and GTP hydrolysis triggers IF2 release from the 70S initiation complex. The next question we have addressed is why multiple copies of L12 dimer are needed on the ribosome. For this purpose, we created a pure E. coli strain JE105, where the terminal part of rplJ gene coding for the binding site of one L12 dimer on protein L10 was deleted in the chromosomal locus. Using ribosomes with single L12 dimer we have observed that the rate of the initiation and elongation involving IF2 and EF-G gets most compromised, which in turn decreases the growth rate of the bacteria.  This study also indicates that L12 can interact with different GTPase factors in a specialized manner. Lastly, we have developed an application making advantage of the multiple L12 dimers on the ribosome. By inserting a (His)6-tag at the C-terminus of the L12 protein we have created a novel E. coli strain (JE28), where all ribosomes are tetra-(His)6-tagged. Further, we have developed a single step method for purification of the active (His)6-tagged ribosomes from JE28.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 848
Keyword
Ribosome, protein synthesis, L12 dimer, initiation, elongation, G factors, and His-tag
National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-157198 (URN)978-91-554-8144-5 (ISBN)
Public defence
2011-10-06, B41, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-09-15 Created: 2011-08-19 Last updated: 2011-11-03Bibliographically approved
2. Versatile Implementations of an Improved Cell-Free System for Protein Biosynthesis: Functional and structural studies of ribosomal protein L11 and class II release factor RF3. Novel biotechnological approach for continuous protein biosynthesis
Open this publication in new window or tab >>Versatile Implementations of an Improved Cell-Free System for Protein Biosynthesis: Functional and structural studies of ribosomal protein L11 and class II release factor RF3. Novel biotechnological approach for continuous protein biosynthesis
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Mångsidig Användning av ett Förbättrat Cell-Fritt System för Proteinbiosyntes : Funktionella och strukturella studier av ribosomalt protein L11 och klass II release faktor RF3. Ny bioteknologisk metod för kontinuerlig proteinbiosyntes
Abstract [en]

Advances in genetics, proteomics and chromatography techniques have enabled the successfully generation of a cell-free bacterial translation system composed of highly pure and active components. This system provided an ideal platform for better elucidating the mechanism of each individual step of the prokaryotic protein biosynthesis and the function of the translation factors involved in the process.

In doing so, we have discovered that the N-terminal domain or complete deletions of the ribosomal protein L11 reduced the termination efficiency of RF1 on cognate stop codons by four to six folds. The L11 deletions also conferred a two folds decrease in the missense error suggesting the increased nonsense termination accuracy of RF2 by two folds, which would clarified previous in vivo observations.

The versatility of the cell-free system has provided the additional possibility to study the effects of class II release factor RF3 mutations in mediating fast dissociation of class I release factors RF1 and RF2 from the post-termination ribosome complexes. The results show a series of mutations within RF3 conferring considerable reduction of the class I release factors recycling rate. These observations together with sequence alignment studies suggest the possible location on RF3 of the class I release factors interaction site.

In addition, the utilization of the cell-free system has made it possible to develop a new biotechnological approach for continuous production of polypeptides, based on gel filtration chromatography. The pilot trials have so far resulted in a six fold production increase of the MFTI test peptide compared to the conventional batch method.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 94 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 144
Keyword
Molecular biology, Protein synthesis, translation termination, ribosome, release factor, cell-free system, missense error, gel filtration, affinity chromatography, Molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-6325 (URN)91-554-6455-6 (ISBN)
Public defence
2006-02-16, Room B22, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2006-01-27 Created: 2006-01-27 Last updated: 2012-03-08Bibliographically approved

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