Künstlich gespaltene Inteine für die Protein-Semisynthese - Charakterisierung des Ssp DnaB Inteins und Modifikation des Ionenkanals OmpF mit Hilfe des Psp-GBD Pol Inteins

Gespaltene Inteine sind leistungsfähige Werkzeuge zur Knüpfung nativer Peptidbindungen zwischen Polypeptidsequenzen. Die von ihnen vermittelte Protein trans-Splei߬reaktion beginnt zunächst mit der Assoziation der Fragmente des gespaltenen Inteins, die Teile von zwei separaten Fusionsproteinen sind....

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Bibliographic Details
Main Author: Brenzel, Steffen
Contributors: Mootz, Henning (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:German
Published: Philipps-Universität Marburg 2009
Chemie
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Table of Contents: Split inteins are versatile tools for the ligation of polypeptide sequences via native peptide bonds. The protein trans-splicing reaction begins with the association of the intein fragments, which are parts of two separate polypeptide sequences. The splicing competent intein complex then mediates its own excision out of the precursor protein and concomitantly links the fused N- and C-extein sequences. The specific intein fragment association makes this ligation of two separately prepared protein fragments a highly chemoselective reaction. Besides several naturally occurring examples the growing number of artificially split inteins becomes more and more important. To expand the scope of protein trans-splicing the Ssp DnaB intein, split after position 104 and lacking the endonuclease domain, was characterized in vitro under native conditions. The importance of assisted association via rapamycin-induced hetero¬dimerzation of the fused FKBP and FRB domains for the protein splicing activity was a major issue in this work. It was found that the intein fragments exhibited pronounced affinity resulting in spontaneous protein trans-splicing even without induced dimerization. Kinetic analyses revealed that the FKBP-FRB association had no positive impact on reaction kinetics and splicing yields. Therefore, the Ssp DnaB intein was the first artificially split intein with spontaneous in vitro splicing activity, which was independent of denaturation and subsequent renaturation of the mixed intein fragments. This can be very useful for applications under completely native conditions to preserve the activity of proteins, which are difficult to refold. In comparison, the rapamycin-induced protein trans-splicing reaction of the Sce VMA intein possessed a similar kinetic potential. A biphasic reaction progress, however, indicated a more complex coupling of the individual reaction steps. In an advanced application, the protein trans-splicing reaction was used for the semisynthesis of the porin OmpF, which is integrated into the outer membrane of E. coli. The 340 amino acids comprising ion channel was reassembled from two recombinantly produced and solubilized protein fragments by means of the artificially split Psp-GBD Pol intein under 6 M urea conditions. The trimeric structure of the OmpF porin with native amino acid sequence could be reconstituted by refolding. Electrophysiological analyses in a planar lipid bilayer (black lipid membrane) proved the native activity of the reassembled OmpF ion channel. Based on these findings, a strategy for the production of a semisynthetic porin was developed combining cysteine conjugation, protein trans-splicing, and refolding. The incor¬poration of single cysteine residues into the N- and C-terminal OmpF fragments and the construction of a cysteine-free intein mutant facilitated the conjugation of a benzo-18-crown-6 building block with the N terminal OmpF fragment (K16C). Protein trans-splicing produced the full-length sequence of the semisynthetic OmpF-derivative, which gave the trimeric pore upon refolding. The crown ether modification inside the pore lumen provoked a reduced channel conductance. This reaction sequence allows for the chemo- and regioselective cysteine conjugation within one fragment independent of more cysteine-residues within the complementary sequence. This in combination with the click reaction can open up new ways for the protein engineering of integral membrane proteins. In conclusion, new approaches could be established, which demonstrate and extend the potential of split inteins for the selective chemical modification of proteins.