The synthesis of non-canonical amino acids and their incorporation into proteins that allow the expansion of functionality and the improvement of protein properties are becoming increasingly important. In this diploma thesis, we synthesized the unnatural amino acid (S)-2-amino-3-(4-prop-2-ynoxyphenyl)propanoic acid with a built-in alkyne moiety. The chemical synthesis was carried out in three steps starting from L-Boc-tyrosine.
In the further process of preparing recombinant proteins N-terminal domain of the protein RimM (ribosome maturation factor) from the organism Thermus thermophilus, BRP (bleomycin resistance protein) from the organism Streptoalloteichus hindustanus and lectin from the organism Oscillatoria agardhii, we attempted to incorporate the synthesized unnatural amino acid into the desired position in the protein. We used the method of incorporation based on suppression of the stop codon (TAG). We expressed protein sequences using the pET28(+) and pEVOL-pRpF vectors in E. coli. By a complementary strategy, for comparison, we introduced an alkyne handle into the protein using bioconjugation approach with NHS alkyne functionalized reagents, resulting in proteins with installed alkyne at different positions. We tested the chemical reactivity of the introduced alkyne handle in selected proteins using the Glaser-Hay bioconjugation and the terminal alkyne reaction with cysteine.
|
