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Protein–DNA Recognition: In Vitro Evolution and Characterization of DNA-Binding Proteins
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

DNA-recognizing proteins are involved in a multitude of important life-processes. Therefore, it is of great interest to understand the underlying mechanisms that set the rules for sequence specific protein–DNA interactions. Previous attempts aiming to resolve these interactions have been focused on naturally occurring systems. Due to the complexity of such systems, conclusions about structure–function relationship in protein–DNA interactions have been moderate.

To expand the knowledge of protein–DNA recognition, we have utilized in vitro evolution techniques. A phage display system was modified to express the DNA-binding, helix-turn-helix protein Cro from bacteriophage λ. A single-chain variant of Cro (scCro) was mutated in the amino acid residues important for sequence-specific DNA-binding. Three different phage-libraries were constructed.

Affinity selection towards a synthetic ORas12 DNA-ligand generated a consensus motif. Two clones containing the motif exhibited high specificity for ORas12 as compared to control ligands. The third library selection, based on the discovered motif, generated new protein variants with increased affinity for ORas-ligands. Competition experiments showed that Arg was important for high affinity, but the affinity was reduced in presence of Asp or Glu. By measuring KD values of similar variant proteins, it was possible to correlate DNA-binding properties to the protein structure.

mRNA display of scCro was also conducted. The system retained the wild-type DNA-binding properties and allowed for functional selection of the mRNA–scCro fusion. Selected species was eluted and the gene encoding the scCro was recovered by PCR.

The two in vitro selection methods described in this thesis can be used to increase the knowledge of the structure–function relationship regarding protein–DNA recognition. Furthermore, we have also shown that new helix-turn-helix proteins exhibiting novel DNA-binding specificity can be constructed by phage display. The ability to construct proteins with altered DNA-specificity has various important applications in molecular biology and in gene therapy.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2004. , p. 50
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1104-232X ; 980
Keywords [en]
Biochemistry, In vitro evolution, Phage display, mRNA display, Cro, DNA recognition, repressor
Keywords [sv]
Biokemi
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-4269ISBN: 91-554-5973-0 (print)OAI: oai:DiVA.org:uu-4269DiVA, id: diva2:164625
Public defence
2004-05-26, Room B21, BMC, Husargatan 3, Uppsala, 10:15
Opponent
Supervisors
Available from: 2004-05-05 Created: 2004-05-05Bibliographically approved
List of papers
1. Functional expression and affinity selection of single-chain Cro by phage display: isolation of novel DNA-binding proteins
Open this publication in new window or tab >>Functional expression and affinity selection of single-chain Cro by phage display: isolation of novel DNA-binding proteins
2000 In: Protein Engineering, Vol. 13, no 7, p. 519-526Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91779 (URN)
Available from: 2004-05-05 Created: 2004-05-05Bibliographically approved
2. Functional properties of in vitro evolved helix-turn-helix proteins: a structural motif for specific recognition of an unnatural DNA ligand
Open this publication in new window or tab >>Functional properties of in vitro evolved helix-turn-helix proteins: a structural motif for specific recognition of an unnatural DNA ligand
Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-91780 (URN)
Available from: 2004-05-05 Created: 2004-05-05Bibliographically approved
3. mRNA-display of DNA-binding helix-turn-helix protein
Open this publication in new window or tab >>mRNA-display of DNA-binding helix-turn-helix protein
Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-91781 (URN)
Available from: 2004-05-05 Created: 2004-05-05Bibliographically approved

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