Implantation of Post‐translational Tyrosylprotein Sulfation into a Prokaryotic Expression System

Lu, Lu Yi; Chen, Bo-Han; Wu, Jennifer Yun Shin; Wang, Chen Chu; Chen, Da Huang; Yang, Yuh‐Shyong

doi:10.1002/cbic.201000540

Cited by 7 publications

(14 citation statements)

References 28 publications

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“…It has been shown that coexpression of the enzyme system and the protein substrate can be achieved in the same prokaryotic expression system and the sulfated protein is produced simultaneously. 25 This system can be very useful for both academic researchers and the future industrial applications to produce desired properly sulfate-modified proteins.…”

Section: Resultsmentioning

confidence: 99%

“… 24 Another method for producing biologically active sulfated proteins has been developed by implanting an artificial PTS system in E. coli . 25 This prokaryotic sulfated protein-generating system involves the coexpression of two plasmids: one pertaining to the expression of the PTS system and the other pertaining to the expression of the target protein. The implementation of this system requires only standard molecular biology techniques; however, the expression of the sulfated protein may have limitations similar to those described for the expanded genetic code method.…”

Section: Introductionmentioning

confidence: 99%

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

Wang

Chen

et al. 2018

ACS Omega

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Wang

Chen

et al. 2018

ACS Omega

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“…In a cell system, radioactive inorganic sulfate can be activated by PAPS synthetase ( Figure 1 ) to form radioactive PAPS. A similar transformation was also used to produce radioactive PAPS in vitro [ 72 ] for the sulfotransferase assay and for the production of organic sulfates. Using radioactive isotope labeling at 35 S and 14 C, about 1% sulfated tyrosine residues were estimated in fly proteins [ 73 ].…”

Section: Methods For the Tpst Assay And Detection Of Ptsmentioning

confidence: 99%

Tyrosine Sulfation as a Protein Post-Translational Modification

et al. 2015

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Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS) is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiological conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST) through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biological sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochemical information including the activity and the preparation of TPST, methods for the determination of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochemical and physiological reactions.

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“…However, compared with the development of gene chips, proteome microarray development is less mature because the techniques used to process proteins are more complex than the corresponding methods used for nucleic acids, and this includes the purification technology, chip fabrication, and analysis approaches. Figure illustrates PTS on a proteome microarray by using a 3′-phosphoadenosine 5′-phosphosulfate (PAPS) synthase-coupled tyrosylprotein sulfotransferase (TPST) catalysis system that involves in situ PAPS generation and TPST catalysis (PTS coupled-enzyme system) …”

mentioning

confidence: 99%

“…Figure 1 illustrates PTS on a proteome microarray by using a 3′-phosphoadenosine 5′-phosphosulfate (PAPS) synthase-coupled tyrosylprotein sulfotransferase (TPST) catalysis system that involves in situ PAPS generation and TPST catalysis (PTS coupled-enzyme system). 25 ■ EXPERIMENTAL SECTION Preparation of E. coli K12 Proteome Microarrays. The high-throughput protein expression, protein purification, and protein printing methods used here were modified from published procedures.…”

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confidence: 99%

High-Throughput Screening of Sulfated Proteins by Using a Genome-Wide Proteome Microarray and Protein Tyrosine Sulfation System

Huang

Chen

et al. 2017

Anal. Chem.

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Protein tyrosine sulfation (PTS) is a widespread posttranslational modification that induces intercellular and extracellular responses by regulating protein-protein interactions and enzymatic activity. Although PTS affects numerous physiological and pathological processes, only a small fraction of the total predicted sulfated proteins has been identified to date. Here, we localized the potential sulfation sites of Escherichia coli proteins on a proteome microarray by using a 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase-coupled tyrosylprotein sulfotransferase (TPST) catalysis system that involves in situ PAPS generation and TPST catalysis. Among the 4256 E. coli K12 proteins, 875 sulfated proteins were identified using antisulfotyrosine primary and Cy3-labeled antimouse secondary antibodies. Our findings add considerably to the list of potential proteins subjected to tyrosine sulfation. Similar procedures can be applied to identify sulfated proteins in yeast and human proteome microarrays, and we expect such approaches to contribute substantially to the understanding of important human diseases.

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Implantation of Post‐translational Tyrosylprotein Sulfation into a Prokaryotic Expression System

Cited by 7 publications

References 28 publications

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

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

Tyrosine Sulfation as a Protein Post-Translational Modification

High-Throughput Screening of Sulfated Proteins by Using a Genome-Wide Proteome Microarray and Protein Tyrosine Sulfation System

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