Preparation of Tyrosylprotein Sulfotransferases for In Vitro One-Pot Enzymatic Synthesis of Sulfated Proteins/Peptides

Abstract

Protein tyrosine sulfation (PTS), catalyzed by membrane-anchored tyrosylprotein sulfotransferase (TPST), is one of the most common post-translational modifications of secretory and transmembrane proteins. PTS, a key modulator of extracellular protein–protein interactions, accounts for various important biological activities, namely, virus entry, inflammation, coagulation, and sterility. The preparation and characterization of TPST is fundamental for understanding the synthesis of tyrosine-sulfated proteins and for studying PTS in biology. A sulfated protein was prepared using a TPST-coupled protein sulfation system that involves the generation of the active sulfate 3′-phosphoadenosine-5′-phosphosulfate (PAPS) through either PAPS synthetase (PAPSS) or phenol sulfotransferase. The preparation of sulfated proteins was confirmed through radiometric or immunochemical assays. In this study, enzymatically active Drosophila melanogaster TPST (DmTPST) and human TPSTs (hTPST1 and hTPST2) were expressed in Escherichia coli BL21(DE3) host cells and purified to homogeneity in high yield. Our results revealed that recombinant DmTPST was particularly useful considering its catalytic efficiency and ease of preparation in large quantities. This study provides tools for high-efficiency, one-step synthesis of sulfated proteins and peptides that are useful for further deciphering the mechanisms, functions, and future applications of PTS.

Silicon Nanowire Field-Effect Transistor as Biosensing Platforms for Post-Translational Modification

“…The results revealed that in situ PTS could be achieved through coupled enzyme treatment, and this was used as supporting data for the following experiment in which in situ PTS was catalyzed on the pSNWFET surface. method that can be used to determine the sulfated protein [28]. The PTS reaction monitored through ELISA was similar to that described in Scheme 2 for PTS on the pSNWFET, with minor modifications.…”

Chemical Methods for N‐ and O‐Sulfation of Small Molecules, Amino Acids and Peptides

“…The sulfate group is a ubiquitous post‐translational modification that accounts for approximately 1 % of all known epigenetic markers, and plays a vital role in a variety of biological processes, including protein–protein and oligosaccharide interactions …”

“…Methods to prepare sulfated proteins have focussed on the sulfated amino acid sY, with very limited examples of sS and sT . The pre‐installation of a sulfate group onto Y is possible, using alternative protecting group methods, but with the added complication of the instability of sY to solid‐phase peptide cleavage.…”

“…The sulfate group is a ubiquitous post-translational modification that accounts for approximately 1 % of all known epigenetic markers, and plays a vital role in a variety of biological processes, including protein-protein and oligosaccharide interactions. [1][2][3][4][5][6][7][8][9] Yet, sulfoproteins have been overlooked in favour of more common phosphorylated proteins, thus limiting our exploration of their function. Recent developments in ultra-high-resolution mass spectrometry have revealed a m/z difference of 9.5 mDa between kinase phosphorylation and sulfation.…”

“…Methods to prepare sulfated proteins have focussed on the sulfated amino acid sY, [2][3][4][5] with very limited examples of sS and sT. [14,15] The pre-installation of a sulfate group onto Y is possible, using alternative protecting group methods, [14,[16][17][18][19][20][21][22][23][24][25][26] but with the added complication of the instability of sY to solidphase peptide cleavage. Chemical methods have various merits and drawbacks, but do allow for pinpoint accuracy in installing a sulfate group.…”