Accelerated Protein Synthesis via One-Pot Ligation-Deselenization Chemistry

Abstract

Here, we describe the development of a highly efficient one-pot ligationdeselenization technology at aspartate and glutamate that enables the synthesis of polypeptides and proteins on unprecedented timescales. The power of the methodology is showcased through the rapid assembly of three thrombininhibiting tick-derived proteins as well as the synthesis of the 21 kDa homodimeric selenoprotein K. This work lays the foundation for the facile synthesis of a range of bioactive polypeptides and proteins in the future. HIGHLIGHTSRapid one-pot ligationdeselenization at b-selenoaspartate and g-selenoglutamate Methodology enables chemical protein synthesis on unprecedented timescales Synthesis of Selenoprotein K through chemoselective deselenization of b-selenoaspartate Synthesis, purification, and quantification of thrombin inhibitory proteins in 3 hr Mitchell et al., Chem 2, 703-715 May 11, 2017 ª 2017 Elsevier Inc. http://dx. SUMMARYPeptide ligation chemistry has revolutionized protein science by facilitating access to synthetic proteins. Here, we describe the development of additive-free ligation-deselenization chemistry at b-selenoaspartate and g-selenoglutamate that enables the generation of native polypeptide products on unprecedented timescales. The deselenization step is chemoselective in the presence of unprotected selenocysteine, which is highlighted in the synthesis of selenoprotein K. The power of the methodology is also showcased through the synthesis of three tick-derived thrombin-inhibiting proteins, each of which were assembled, purified, and isolated for biological assays within a few hours. The methodology described here should serve as a powerful means of accessing synthetic proteins, including therapeutic leads, in the future.

Selenium and Selenocysteine in Protein Chemistry

“…[81] Similarly,( b-Se)-Asp and (g-Se)-Glu were incorporated in the synthesis of selenoprotein Ka nd three thrombin-inhibiting proteins,i n which one-pot ligation-deselenization gave the native proteins. [82] Sec-mediated NCL has been used in the assembly of diselenide- [83] and selenosulfide-containing analogues of bovine pancreatic trypsin inhibitor (BPTI) [73] and the C-terminal segment of ribonucleotide reductase, [72] among others.I n addition, Sec-mediated expressed-protein ligation (EPL), [84] in which the majority of the protein is recombinantly expressed fused to an intein, converted into at hioester,a nd ligated to ap eptide containing Cys or Sec,l ed to the semisynthesis of Sec-containing variants of RNase A, [74] the natural selenoprotein TR3, [85] and the copper metalloprotein azurin. [86] Azurin is ap articularly good candidate for EPL because its active-site Cys112 is located in the C-terminal portion of the protein, and the C112U variant binds copper as well as the original protein does.…”

Selen und Selenocystein in der Proteinchemie

“…[80] Mit einem Prolylselenoesterpeptid als reaktiverem Acyldonor in der Pro-Cys-Ligation, die unter klassischen Bedingungen extrem langsam vonstatten geht, läuft die NCL bedeutend leichter ab. [82] Die Sec-vermittelte NCL wurde unter anderem eingesetzt, um Diselenid- [83] und Selensulfid-haltige Analoga des Rinder-Trypsininhibitors (BPTI) [73] sowie des C-terminalen Segments von Ribonucleotidreduktase [72] zu erzeugen. [82] Die Sec-vermittelte NCL wurde unter anderem eingesetzt, um Diselenid- [83] und Selensulfid-haltige Analoga des Rinder-Trypsininhibitors (BPTI) [73] sowie des C-terminalen Segments von Ribonucleotidreduktase [72] zu erzeugen.…”

“…[97] Im Gegensatz dazu ist die Deselenierung von Sec zu Ala chemoselektiv und hat keine Auswirkungen auf ungeschützte Cys-Reste (Abbildung 10 a). [82,[98][99][100] Darüber hinaus ergibt die Methylierung von Homoselenocystein nach NCL, [101] die in [82,[98][99][100] Darüber hinaus ergibt die Methylierung von Homoselenocystein nach NCL, [101] die in …”

“…[81] Auf ähnliche Weise wurden (b-Se)-Asp und (g-Se)-Glu bei der Synthese von Selenoprotein Ku nd drei Thrombin-inhibierendenP roteinen gefolgt von einer Ein-Topf-Ligation/Deselenierung eingebaut. [82] Die Sec-vermittelte NCL wurde unter anderem eingesetzt, um Diselenid- [83] und Selensulfid-haltige Analoga des Rinder-Trypsininhibitors (BPTI) [73] sowie des C-terminalen Segments von Ribonucleotidreduktase [72] zu erzeugen. Zusätzlich dazu gelang mit der Sec-vermittelten Ligation exprimierter Proteine (EPL), [84] bei der die Mehrheit der Proteine als Intein-Fusionen rekombinant exprimiert, in Thioester überführt und dann mit einem Cys-oder Sec-haltigen Peptid ligiert wird, die Semisynthese Sec-haltiger Va rianten der RNase A, [74] des natürlichen Selenoproteins TR3 [85] und des Kupfer-Metalloproteins Azurin.…”

“…[79] ¾hnlich wie die Desulfurierung wurde auch der Deselenierungsansatz auf andere Aminosäuren (Pro,Phe,Leu, Asp und Glu) mit künstlich eingeführten Selenolfunktionen erweitert (Abbildung 10 b). [82,[98][99][100] Darüber hinaus ergibt die Methylierung von Homoselenocystein nach NCL, [101] die in…”