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Enzymatische Ligation von Peptiden, Peptidnucleinsäuren und Proteinen
| Content Provider | Semantic Scholar |
|---|---|
| Author | Pritz, Stephan |
| Copyright Year | 2009 |
| Abstract | Peptides and proteins are important research objects in biochemical research. Therefore, several ligation methods to couple unprotected, purified peptide sequences in aqueous media have been developed during the last years. At a special interest in this case is the bacterial transpeptidase sortase A. This enzyme couples proteins in vivo to the bacterial peptidoglycan by cleavage at a LPXTG-recognition motif between threonine and glycine and subsequent transfer to an oligoglycin nucleophile. In order to investigate the enzymatic activity, a simple HPLC-based assay was established in this work. Results obtained with model peptides were used for the assembly of a soluble receptor. A key step was the sortase-mediated ligation of the folded receptor N-terminus (expressed in E. coli) to the 3-loop-construct. The resulting receptor mimic of 23 kDa was homogeneous according to HPLC and MS. It showed specific binding to natural peptide ligands of the CRF1-receptor with high affinity. Furthermore, it could be shown that sortase is usable for selective protein labeling. For this purpose, a fluorescence label was attached C-terminally to the 50 kDa protein NEMO. As a further example of sortase-mediated ligation served the synthesis of PNA-CPPconjugates. The use of an excess of the peptide and dialyzing away the small leaving group proved to be very effective and coupling yields up to 94% could be achieved. The biological activity of the CPP-PNA-conjugates could be shown by uptake studies in cells. |
| File Format | PDF HTM / HTML |
| DOI | 10.18452/15881 |
| Alternate Webpage(s) | https://edoc.hu-berlin.de/bitstream/handle/18452/16533/pritz.pdf?isAllowed=y&sequence=1 |
| Alternate Webpage(s) | http://edoc.hu-berlin.de/dissertationen/pritz-stephan-2008-05-23/PDF/pritz.pdf |
| Alternate Webpage(s) | https://doi.org/10.18452/15881 |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
