TY - BOOK

T1 - Split-click protein chemistry

AU - Kofoed, Christian

PY - 2018

Y1 - 2018

N2 - Proteases are among the largest families of enzymes, catalyzing the hydrolysis of peptide bonds. However, proteases remain a class of enzymes that are difficult-to-express, due to their cell toxicity. Alternatively proteases could be expressed as two inactive halves that can be stitched together using chemistry. In this thesis, the focus will be on using copper(i)-catalyzed azide-alkyne cycloaddition, also popularly known as click chemistry, as a ligation method to produce a reconstituted enzyme from two inactive halves. This method was named split-click protein chemistry. As a proof-of-concept, two split-fragments of the model enzyme tobacco etch virus protease were produced by recombinant expression, allowing for the incorporation of noncanonical amino acids functionalized with azide or alkyne moieties. Residue-specific replacement of methionine was used for the incorporation of the two methionine analogues azidohomoalanine and homopropargylglycine. Amber stop codon suppression was used for the incorporation of two lysine derivatives, functionalized with azide or alkyne moieties. The incorporation of the clickable noncanonical amino acids allowed for the ligation of the splitfragments. In this way, clicking the azide and alkyne moieties was used to generate loop mimics. One the ligation products was investigated further and shown to be compatible with enzyme activity, as the clicked protein had maintained biological activity.

AB - Proteases are among the largest families of enzymes, catalyzing the hydrolysis of peptide bonds. However, proteases remain a class of enzymes that are difficult-to-express, due to their cell toxicity. Alternatively proteases could be expressed as two inactive halves that can be stitched together using chemistry. In this thesis, the focus will be on using copper(i)-catalyzed azide-alkyne cycloaddition, also popularly known as click chemistry, as a ligation method to produce a reconstituted enzyme from two inactive halves. This method was named split-click protein chemistry. As a proof-of-concept, two split-fragments of the model enzyme tobacco etch virus protease were produced by recombinant expression, allowing for the incorporation of noncanonical amino acids functionalized with azide or alkyne moieties. Residue-specific replacement of methionine was used for the incorporation of the two methionine analogues azidohomoalanine and homopropargylglycine. Amber stop codon suppression was used for the incorporation of two lysine derivatives, functionalized with azide or alkyne moieties. The incorporation of the clickable noncanonical amino acids allowed for the ligation of the splitfragments. In this way, clicking the azide and alkyne moieties was used to generate loop mimics. One the ligation products was investigated further and shown to be compatible with enzyme activity, as the clicked protein had maintained biological activity.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122252331005763

M3 - Ph.D. thesis

BT - Split-click protein chemistry

PB - Department of Chemistry, Faculty of Science, University of Copenhagen

ER -