Gerichtete Proteinevolution als Werkzeug zur Generierung funktionell und strukturell neuartiger Proteine

Proteine mit neuartiger oder verbesserter Funktionalität sind in der Industrie und der Grundlagenforschung von großem Interesse. Im Rahmen dieser Doktorarbeit wurden zwei Verfahren der gerichteten Proteinevolution, das Hefe-2-Hybridsystem und das Phagen-Display, verwendet und auf die Anforderungen f...

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Bibliographic Details
Main Author: Garbe, Daniel
Contributors: Mootz, Henning D. (Professor Dr.) (Thesis advisor)
Format: Dissertation
Published: Philipps-Universität Marburg 2009
Online Access:PDF Full Text
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Table of Contents: Proteins bearing new or improved functionalities are very important in industry and basic research. This PhD thesis deals with two directed protein evolution methods, the yeast-2-hybrid system and phage display, which were adopted to the needs of two different proteins. The aim of the first project was to develop a protein that can be artificially switched by an organic molecule. This molecule should bind in a newly created cavity established in the hydrophobic core of the model protein, the SH3 domain of the c-Abl tyrosine kinase. To this end the small hydrophobic core amino acid Ala4 was replaced in a first step by the bulky Val and Phe. Following the adaptation of the protein folding to this steric constraint, a back-mutation was intended to reveal a new cavity for binding of the organic molecule. Unlike the Phe mutant, the Val substitution was tolerated by the protein fold and allowed its biochemical characterisation by CD-spectroscopy and a peptide ligand binding assay. A protein evolution approach using a yeast-2-hybrid selection system was developed to identify compensating mutations in the A4F mutant in order to stabilize again the hydrophobic core. Fusion constructs of the B42 activator domain and the DNA binding domain LexA with the peptide ligand and the SH3 domain were created. An interaction between the SH3 domain and the ligand was necessary to promote transcription of the reporter genes LEU2 and lacZ. Two libraries of the SH3 domain were prepared on the DNA-level, one by epPCR and the other by focussed random mutagenesis of the five remaining amino acids of the hydrophobic core (L16, I18, L24, V47 and I52). Genetically selected yeast colonies were further characterised. However, it was noticed that a large number of the selected yeast cells were false positives. Hence the reorganisation of the hydrophobic core could not be accomplished. In the second project a phage display scheme to select for split inteins with improved activity was developed for the first time. Inteins are internal proteins that excise themselves in an autocatalytic fashion from a precursor protein with concomitant linkage of two flanking polypeptide chains (N- and C-extein). This reaction is referred to as protein splicing. In split inteins the N- and C-extein fragments have to associate before the splicing reaction. The object of this project was the DnaB intein from Synecchocystis sp. PCC6803, which was artificially split either at position 11 or at position 104. Protein splicing was shown between an IntN(104) fusion protein and an M13-Phage presenting the complementary IntC fragment. Subsequently, the IntN(104) fusion protein was immobilisied on streptavidin beads via its N-terminal streptavidin binding peptide (SBP). The resulting splice product (SBP-phage) could be specifically eluted from the beads with biotin. Following this protocol it was possible to enrich the hybrid-phage from a 1:10,000 mixture with a control-phage incompetent for protein splicing. After five rounds of biopanning an enrichment factor of 1,250 was obtained. In experiments with the DnaB intein split at position 11 a fairly different protocol was used. In this case, the N-terminal modification desthiobiotin of a synthetic IntN(11) peptide was transferred to the splice product, which could then be immobilized on streptavidin beads. By this protocol, an enrichment of the hybrid-phage from a 1:10,000 mixture with a control-phage by an average factor of 290 was accomplished. Finally the influence of the amino acid at position +2 of the C-extein on the splicing activity of the DnaB intein split at position 104 was examined using purified proteins. To this end a saturated mutagenesis with a degenerated oligonucleotide was applied at this position. Besides the naturally occurring isoleucine, alanine showed a positive influence, whereas Pro caused an inhibitory effect on the progress of the in vitro protein trans-splicing reaction.