TPST1 Human, sf9
Description
Introduction to Tyrosylprotein Sulfotransferase 1
Tyrosylprotein Sulfotransferase 1, commonly known as TPST1, belongs to the protein sulfotransferase family and plays a crucial role in the post-translational modification of proteins through tyrosine sulfation . This enzyme catalyzes the transfer of a sulfonate group from 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) to tyrosine residues in target proteins, forming tyrosine O-sulfate ester groups while releasing the desulfonated 3'-phosphoadenosine-5'-phosphate . Protein tyrosine sulfation (PTS) represents one of the most common post-translational modifications of secretory and transmembrane proteins, serving as a key modulator of extracellular protein-protein interactions .
TPST1 is also known by several alternative names including EC 2.8.2.20, TPST-1, Transport And Golgi Organization 13 Homolog A (Drosophila), Tyrosylprotein Sulfotransferase-1, and TANGO13A . The enzyme has been extensively studied due to its involvement in various important biological activities including virus entry, inflammation, coagulation, and sterility .
Production and Characterization of TPST1 Human, sf9
TPST1 Human, sf9 refers specifically to human TPST1 that has been recombinantly expressed in Sf9 insect cells using a baculovirus expression system. This expression system was chosen due to its ability to produce eukaryotic proteins with appropriate post-translational modifications, which are crucial for the proper folding and function of many human proteins .
Expression System and Production Method
The recombinant TPST1 is produced in Sf9 insect cells infected with baculovirus carrying the TPST1 gene . This expression system offers several advantages over prokaryotic expression systems, particularly for complex human proteins that require glycosylation and proper disulfide bond formation. The baculovirus-insect cell system closely mimics the post-translational processing that occurs in mammalian cells, resulting in a more native-like recombinant protein .
Physical and Biochemical Properties
TPST1 Human, sf9 possesses distinct physical and biochemical characteristics that are summarized in Table 1.
Table 1: Physical and Biochemical Properties of TPST1 Human, sf9
The difference between theoretical molecular mass (40.6 kDa) and the observed migration pattern on SDS-PAGE (40-57 kDa) can be attributed to the glycosylation of the protein, which is a common characteristic of proteins expressed in eukaryotic systems .
Primary Structure and Domain Organization
The recombinant TPST1 Human, sf9 represents a truncated version of the native enzyme, lacking the N-terminal cytoplasmic domain and the transmembrane domain. The construct includes residues 26-370 of the full-length human TPST1, focusing primarily on the catalytic domain of the enzyme .
Human TPST1 has 370 amino acids with a predicted molecular mass of 42.2 kDa for the full-length protein . The native enzyme possesses a type II transmembrane topology with a short eight-residue N-terminal cytoplasmic domain, a 17-residue transmembrane domain, and a luminal catalytic domain . This topology is consistent with the enzyme's localization in the trans-Golgi network, where it catalyzes the sulfation of tyrosine residues in proteins destined for secretion or membrane insertion .
Amino Acid Sequence
The complete amino acid sequence of TPST1 Human, sf9 is presented in Table 2, which provides the primary structure of this recombinant protein.
Table 2: Amino Acid Sequence of TPST1 Human, sf9
| Sequence Position | Amino Acid Sequence |
|---|---|
| 1-50 | ADPQHAMECH HRIEERSQPV KLESTRTTVR TGLDLKANKT FAYHKDMPLI |
| 51-100 | FIGGVPRSGT TLMRAMLDAH PDIRCGEETR VIPRILALKQ MWSRSSKEKI |
| 101-150 | RLDEAGVTDE VLDSAMQAFL LEIIVKHGEP APYLCNKDPF ALKSLTYLSR |
| 151-200 | LFPNAKFLLM VRDGRASVHS MISRKVTIAG FDLNSYRDCL TKWNRAIETM |
| 201-250 | YNQCMEVGYK KCMLVHYEQL VLHPERWMRT LLKFLQIPWN HSVLHHEEMI |
| 251-300 | GKAGGVSLSK VERSTDQVIK PVNVGALSKW VGKIPPDVLQ DMAVIAPMLA |
| 301-350 | KLGYDPYANP PNYGKPDPKI IENTRRVYKG EFQLPDFLKE KPQTEQVE |
| 351-360 | HHHHHH (C-terminal His-tag) |
This sequence represents the catalytic domain of human TPST1 (residues 26-370 of the full-length protein) with the addition of a six-histidine tag at the C-terminus to facilitate purification .
Sequence Conservation and Homology
Furthermore, substantially higher sequence identity (>90%) is found between human TPST1 and its corresponding enzyme in other mammalian species, suggesting strong evolutionary conservation of this enzyme's function .
Enzymatic Activity and Catalytic Mechanism
TPST1 catalyzes the transfer of a sulfonate group from 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) to specific tyrosine residues in target proteins . The enzyme binds both PAPS as the sulfonate donor and proteins containing target tyrosine residues, facilitating the formation of tyrosine O-sulfate ester groups while generating desulfonated 3'-phosphoadenosine-5'-phosphate as a byproduct .
The catalytic activity of TPST1 is influenced by various factors including pH, divalent cations, and substrate specificity. Compared to TPST2, TPST1 typically displays lower Km and Vmax values for most peptide substrates, suggesting differential substrate specificity between the two isoforms . The activity of TPST1 is characterized by an acidic pH optimum, which aligns with its localization in the trans-Golgi network where an acidic environment prevails .
Physiological Role and Tissue Distribution
TPST1 plays a critical role in the post-translational modification of numerous secretory and transmembrane proteins. Protein tyrosine sulfation mediated by TPST1 significantly influences protein-protein interactions in the extracellular environment, affecting various biological processes including inflammation, coagulation, and viral entry into cells .
The enzyme is widely distributed across human tissues, with TPST activity detected in various cells and organs including the adrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung, pancreas, peripheral leukocytes, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid gland, trachea, and uterus . This broad tissue distribution underscores the fundamental importance of tyrosine sulfation in human physiology.
While both TPST1 and TPST2 are co-expressed in all human tissues examined, their expression levels vary significantly among different tissues, suggesting distinct physiological functions for each isoform .
Research Applications
TPST1 Human, sf9 serves as a valuable research tool for studying protein tyrosine sulfation and its biological significance. The recombinant enzyme enables in vitro sulfation of target proteins, facilitating investigations into the structural and functional consequences of this post-translational modification .
One notable application involves the coupled enzyme methods for generating sulfated proteins and peptides. TPST1 can be used in combination with PAPS synthetase (PAPSS) or phenol sulfotransferase to create a protein sulfation system that produces sulfated proteins for further study . Such sulfated proteins can be detected and characterized using various techniques including radiometric assays, enzyme-linked immunosorbent assay (ELISA), and fluorimetric methods .
Therapeutic Potential
Understanding the mechanisms and functions of protein tyrosine sulfation has significant implications for therapeutic development. Since tyrosine sulfation modulates protein-protein interactions involved in critical biological processes like inflammation and viral entry, TPST1 and its substrates represent potential targets for therapeutic intervention in various diseases .
The availability of recombinant TPST1 Human, sf9 facilitates high-throughput screening of potential inhibitors or activators of the enzyme, which could lead to the development of novel therapeutics targeting tyrosine sulfation-dependent processes .
Product Specs
Q&A
Tyrosylprotein sulfotransferase 1 (TPST1) is a critical enzyme mediating protein tyrosine sulfation, a post-translational modification regulating extracellular protein interactions. Below are structured FAQs addressing key research considerations for working with TPST1 expressed in Sf9 baculovirus systems, organized by experimental complexity and supported by biochemical data.
Intermediate Experimental Challenges
Q3: How to address discrepancies in TPST1 vs. TPST2 substrate specificity?
Substrate preferences arise from structural differences in their catalytic domains:
Solution: Use chimeric enzymes or site-directed mutagenesis to map specificity determinants .
Q4: What controls are essential for TPST1 kinetic assays?
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Negative controls: Omit PAPS or use catalytically inactive mutants (e.g., Cys→Ala substitutions) .
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Positive controls: Include PSGL1-tide, a high-efficiency substrate .
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Buffer considerations: 50 mM HEPES (pH 7.4), 5 mM MnCl₂ for optimal activity .
Advanced Mechanistic Studies
Q5: How do RAF kinase inhibitors cross-react with TPST1?
RAF inhibitors (e.g., RAF265, vemurafenib) inhibit TPST1 via competitive binding:
| Inhibitor | TPST1 IC₅₀ | Primary Target | Structure | Source |
|---|---|---|---|---|
| RAF265 | 6.5 µM | c-RAF kinase | 2-arylaminobenzimidazole | |
| GW458344A | 4.2 µM | c-RAF kinase | Aza-stilbene | |
| Sorafenib | >100 µM | B-RAF kinase | Diarylurea |
Implications: Repurpose RAF inhibitor libraries for TPST1 modulation studies .
Q6: What structural features explain TPST1’s processive sulfation of clustered tyrosines?
Crystal structures reveal:
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A charged substrate-binding groove accommodating acidic residues (e.g., Glu/Asp near Tyr) .
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Dynamic conformational changes enabling sequential sulfation of N-terminal tyrosines .
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Dimerization interfaces (homo-/heterodimers) that enhance processivity.
Technical Troubleshooting
Q7: Why does TPST1 exhibit variable activity in different buffer conditions?
Key factors affecting activity:
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Divalent cations: Mn²⁺ (5 mM) enhances sulfation 2-fold vs. Mg²⁺ .
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Reducing agents: Avoid DTT >1 mM to prevent disulfide bond disruption .
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Storage: Add 0.1% HSA and store at -80°C for long-term stability .
Q8: How to resolve conflicting sulfation data between in vitro and cellular assays?
Potential causes:
Product Science Overview
Tyrosylprotein Sulfotransferase 1 (TPST1) is an enzyme that plays a crucial role in the post-translational modification of proteins. Specifically, it catalyzes the sulfation of tyrosine residues within polypeptides, a process essential for various biological functions. TPST1 belongs to the protein sulfotransferase family and is involved in the formation of tyrosine O-sulfate ester groups .
The recombinant form of TPST1 is produced in Sf9 insect cells using the baculovirus expression system. This method allows for the production of a single, glycosylated polypeptide chain containing 354 amino acids (from 26 to 370 a.a.) with a molecular mass of approximately 40.6 kDa . The recombinant TPST1 is expressed with a 6-amino acid His tag at the C-terminus, which facilitates its purification through chromatographic techniques .
TPST1 is a membrane-bound enzyme localized in the Golgi apparatus. It utilizes 3’-phosphoadenosine-5’-phosphosulfate (PAPS) as the sulfonate donor and binds to proteins with target tyrosine residues to form the tyrosine O-sulfate ester group . The enzyme’s activity is crucial for various biological processes, including the inflammatory response .
The recombinant TPST1 protein is typically supplied as a sterile, filtered colorless solution. It is formulated in phosphate-buffered saline (PBS) with 10% glycerol to enhance its stability . For short-term storage, the protein can be kept at 4°C if used within 2-4 weeks. For long-term storage, it is recommended to store the protein at -20°C, with the addition of a carrier protein such as human serum albumin (HSA) or bovine serum albumin (BSA) to prevent multiple freeze-thaw cycles .
Recombinant TPST1 is widely used in biochemical and pharmaceutical research to study the sulfation of tyrosine residues and its implications in various biological processes. Its high purity and specific activity make it a valuable tool for investigating the role of tyrosine sulfation in protein function and signaling pathways .
