Entwicklung einer neuen Methode zur chemo- und regioselektiven Cys-tag Modifikation von Proteinen mithilfe von gespaltenen Inteinen

The chemo- and regioselective introduction of synthetic moieties into proteins is of fundamental importance in basic protein and biochemical research. Several techniques have been developed for this purpose, which differ in the nature of the synthetic moiety and the selectivity of its incorporation. A general important criterion is thereby that the structural and functional integrity of the protein under investigation is preserved. In the majority of traditional bioconjugate techniques the sulfhydryl group of cysteine residues provides the most important functional target for modifications, due to its unique chemical reactivity and low natural abundance in proteins. However, cysteine modification is strictly regioselective only in proteins with a single cysteine residue and becomes severely limited or even impractical for target proteins containing essential or multiple cysteines. The approach outlined in this work circumvents the problem of regioselectivity by a two-step procedure using a split intein. In a first step, a fusion protein consisting of a C-terminal fragment of a split intein and a short cysteine-containing C-extein sequence (Cys-tag) is modified with a sulfhydryl-reactive probe. In a second step, this labelled Cys-tag is linked to a target protein, which was expressed in fusion with the complementary split intein fragment, with a native peptide bond by protein trans-splicing. Thus, the modification reaction proceeds prior to the attachment and therefore leaves other native cysteine residues in the target protein unaffected. One important prerequisite for this strategy is the absence of a cysteine in the respective intein fragment. In this work the artificially split Ssp DnaB intein was used initially, which showed good yields in time- and temperature-dependant splicing reactions. The introduction of a cysteine residue into the previously cysteine-free C-terminal extein sequence yielded a Cys-tag, that could be efficiently labelled with various fluorophores and biotin. Neither the modified Cys-tag nor the free cysteine itself in close proximity to the active site nucleophile of the intein had an effect on the splicing activity. Moreover, the cysteine-containing E. coli proteins β-lactamase and thioredoxin could be obtained as fusions with the N-terminal DnaB intein fragment and regioselectively labelled with fluorescent probes by retaining enzymatic activity. In addition, the spontaneous association of the complementary intein fragments into its active form could be used for the specific labelling of a crude protein in a cell lysate. In a further development of this approach, an artificially split version of the Mxe GyrA mini-intein could be generated, biochemical characterized and used for the Cys-tag modification of proteins. For the GyrA intein, splice product formation proceeded with slightly slower rates, but with significantly higher yields, compared to the previously used DnaB intein. In addition to the modification with fluorpophores, proteins could also be modified with a polyethylene glycol moiety. The preparation of regioselectively fluorescein-labeled human growth hormone was achieved by the Cys-tag approach in combination with a renaturation protocol. The regioselective fluorescein-modification of the non-ribosomal peptide synthetase TycA served as another example, showing the broad potential of this approach. Modified and purified TycA showed the same enzymatic activity in a product formation assay compared to a recombinantly produced unmodified TycA control protein. Moreover, the new artificially split Mxe GyrA intein was found to be orthogonal to the split Ssp DnaB intein, i.e. both inteins produced their respective splice products independently from each other.