TY - JOUR
T1 - Semisynthesis of an Active Enzyme by Quantitative Click Ligation
AU - Kofoed, Christian
AU - Riesenberg, Stephan
AU - Šmolíková, Jaroslava
AU - Meldal, Morten
AU - Schoffelen, Sanne
PY - 2019/4/17
Y1 - 2019/4/17
N2 - The incorporation of clickable noncanonical amino acids (ncAAs) has proven to an invaluable tool in chemical biology and protein science research. Nevertheless, the number of examples in which the method is used for preparative purposes is extremely limited. We report the synthesis of an active enzyme by quantitative, Cu(I)-catalyzed ligation of two inactive protein halves, expressed and equipped with an azide and alkyne moiety, respectively, through ncAA incorporation. The reported quantitative conversion is exceptional given the large size of the protein fragments and the fact that no linker or excess of either of the polypeptides was used. The triazole bridge formed between the ncAA side chains was shown to effectively mimic a natural protein loop, providing an enzyme with the same activity as its natural counterpart. We envision that this strategy, termed split-click protein chemistry, can be used for the production of proteins that are difficult to express as full-length entities. It also paves the way for the design of new proteins with tailor-made functionalities.
AB - The incorporation of clickable noncanonical amino acids (ncAAs) has proven to an invaluable tool in chemical biology and protein science research. Nevertheless, the number of examples in which the method is used for preparative purposes is extremely limited. We report the synthesis of an active enzyme by quantitative, Cu(I)-catalyzed ligation of two inactive protein halves, expressed and equipped with an azide and alkyne moiety, respectively, through ncAA incorporation. The reported quantitative conversion is exceptional given the large size of the protein fragments and the fact that no linker or excess of either of the polypeptides was used. The triazole bridge formed between the ncAA side chains was shown to effectively mimic a natural protein loop, providing an enzyme with the same activity as its natural counterpart. We envision that this strategy, termed split-click protein chemistry, can be used for the production of proteins that are difficult to express as full-length entities. It also paves the way for the design of new proteins with tailor-made functionalities.
U2 - 10.1021/acs.bioconjchem.9b00110
DO - 10.1021/acs.bioconjchem.9b00110
M3 - Journal article
C2 - 30883092
SN - 1043-1802
VL - 30
SP - 1169
EP - 1174
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
IS - 4
ER -