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Expression and purification of proteins with unnatural aminoacids, and determination of their structure by combination of chemical or photochemical modification and mass spectrometry
Bortel, Tomáš ; Ječmen, Tomáš (advisor) ; Kavan, Daniel (referee)
Residue-specific non-natural amino acid (NNAA) incorporation has become a widely used approach to introduce bioorthogonal groups and therefore functionalities into proteins26 . These functionalities are harnessed through click chemistry, conjugating labelled proteins to affinity or fluorescent probes. A less utilized approach involves probing the effects of bioorthogonal groups on the structure-function relationship of labelled proteins47 . In order to investigate these relationships, there is a need to express large amounts of proteins with maximal incorporation of NNAAs. Here, we employed photo-methionine (pMet), azidohomoalanine (AHA) and homopropargylglycine (HPG) as methionine (Met) surrogates. We investigated the impact of these NNAAs on bacterial growth of prototrophic E. coli BL21 or Met- -auxotrophic E. coli B834 in Met-free MM-M9 medium. We monitored the expression of cytochrome b5 (cyt b5) and MBP-GFP. Using MS and LC-MS based approaches, we determined the NNAA incorporation in these recombinant proteins. Prototrophic E. coli BL21 expressed significantly higher amounts of cyt b5 compared to B834 with pMet and AHA, but the incorporation rates fell sharply after 4 hours. In contrast, Met-auxotrophic B834 expressed smaller amounts of protein, but with incorporation of pMet in 50 - 70% range...
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