Name: Recombinant Mouse L-dopachrome tautomerase (Dct), partial
Brand: CUSABIO
SKU: CSB-EP006562MO1

Product Details

Purity

Greater than 90% as determined by SDS-PAGE.

Target Names

Dct

Uniprot No.

P29812

Research Area

Cell Biology

Alternative Names

Dct; Tyrp-2; Tyrp2; L-dopachrome tautomerase; DCT; DT; EC 5.3.3.12; DOPAchrome conversion factor; DOPAchrome isomerase; DOPAchrome oxidoreductase; L-dopachrome Delta-isomerase; SLATY locus protein; Tyrosinase-related protein 2; TRP-2; TRP2

Species

Mus musculus (Mouse)

Source

E.coli

Expression Region

24-472aa

Target Protein Sequence

QFPRVCMTLDGVLNKECCPPLGPEATNICGFLEGRGQCAEVQTDTRPWSGPYILRNQDDREQWPRKFFNRTCKCTGNFAGYNCGGCKFGWTGPDCNRKKPAILRRNIHSLTAQEREQFLGALDLAKKSIHPDYVITTQHWLGLLGPNGTQPQIANCSVYDFFVWLHYYSVRDTLLGPGRPYKAIDFSHQGPAFVTWHRYHLLWLERELQRLTGNESFALPYWNFATGKNECDVCTDELLGAARQDDPTLISRNSRFSTWEIVCDSLDDYNRRVTLCNGTYEGLLRRNKVGRNNEKLPTLKNVQDCLSLQKFDSPPFFQNSTFSFRNALEGFDKADGTLDSQVMNLHNLAHSFLNGTNALPHSAANDPVFVVLHSFTDAIFDEWLKRNNPSTDAWPQELAPIGHNRMYNMVPFFPPVTNEELFLTAEQLGYNYAVDLSEEEAPVWSTTLS
Note: The complete sequence including tag sequence, target protein sequence and linker sequence could be provided upon request.

Mol. Weight

69.7kDa

Protein Length

Partial

Tag Info

N-terminal 10xHis-SUMO-tagged

Form

Liquid or Lyophilized powder
Note: We will preferentially ship the format that we have in stock, however, if you have any special requirement for the format, please remark your requirement when placing the order, we will prepare according to your demand.

Buffer

If the delivery form is liquid, the default storage buffer is Tris/PBS-based buffer, 5%-50% glycerol.
Note: If you have any special requirement for the glycerol content, please remark when you place the order.
If the delivery form is lyophilized powder, the buffer before lyophilization is Tris/PBS-based buffer, 6% Trehalose.

Reconstitution

We recommend that this vial be briefly centrifuged prior to opening to bring the contents to the bottom. Please reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.We recommend to add 5-50% of glycerol (final concentration) and aliquot for long-term storage at -20°C/-80°C. Our default final concentration of glycerol is 50%. Customers could use it as reference.

Troubleshooting and FAQs

Protein FAQs

Storage Condition

Store at -20°C/-80°C upon receipt, aliquoting is necessary for mutiple use. Avoid repeated freeze-thaw cycles.

Shelf Life

The shelf life is related to many factors, storage state, buffer ingredients, storage temperature and the stability of the protein itself.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.

Lead Time

3-7 business days

Notes

Repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week.

Datasheet & COA

Please contact us to get it.

Description

Dopachrome tautomerase (Dct), also known as tyrosinase-related protein-2 (TRP-2), is a crucial enzyme in the biosynthetic pathway of melanin. It catalyzes the conversion of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a precursor of eumelanins [1]. Dct plays a significant role in melanin synthesis by converting dopachrome to DHICA, contributing to the production of eumelanins [2]. Encoded by the Dct gene, Dct is responsible for the non-decarboxylative tautomerization of L-DOPAchrome to DHICA in the melanin biosynthetic pathway [3].

Beyond melanin synthesis, studies have demonstrated that Dct protects melanocytic cells from damage caused by ultraviolet radiation and reactive oxygen species, thereby enhancing cell viability [4]. Moreover, Dct has been implicated in melanoma cell phenotype-specific interactions, potentially influencing tumor progression [5]. Additionally, Dct has been associated with resistance to certain chemotherapeutic agents in melanoma, underscoring its role in therapeutic implications [6].

The regulation of Dct expression is dynamic and involves factors such as MITF, ER-α, and chromatin remodelers, impacting cell proliferation and senescence in melanocytes [2]. Furthermore, Dct has been linked to retinogenesis misregulation in albinism, highlighting its importance in melanosome function and related disorders [7][8]. The interaction of Dct with caveolin-1 in melanoma cells has been identified as phenotype-specific and potentially involved in tumor progression [5].

References:
[1] G. Costin, J. Valencia, K. Wakamatsu, S. Ito, F. Solano, A. Milacet al., "Mutations in dopachrome tautomerase (dct) affect eumelanin/pheomelanin synthesis, but do not affect intracellular trafficking of the mutant protein", Biochemical Journal, vol. 391, no. 2, p. 249-259, 2005. https://doi.org/10.1042/bj20042070
[2] D. Schwahn, N. Timchenko, S. Shibahara, & E. Medrano, "Dynamic regulation of the human dopachrome tautomerase promoter by mitf, er‐α and chromatin remodelers during proliferation and senescence of human melanocytes", Pigment Cell Research, vol. 18, no. 3, p. 203-213, 2005. https://doi.org/10.1111/j.1600-0749.2005.00229.x
[3] T. Kobayashi, G. Imokawa, D. Bennett, & V. Hearing, "Tyrosinase stabilization by tyrp1 (the brown locus protein)", Journal of Biological Chemistry, vol. 273, no. 48, p. 31801-31805, 1998. https://doi.org/10.1074/jbc.273.48.31801
[4] S. Ainger, X. Yong, S. Wong, D. Škalamera, B. Gabrielli, J. Leonardet al., "dct protects human melanocytic cells from uvr and ros damage and increases cell viability", Experimental Dermatology, vol. 23, no. 12, p. 916-921, 2014. https://doi.org/10.1111/exd.12574
[5] I. Popa, A. Milac, L. Sima, P. Alexandru, F. Pastrama, C. Munteanuet al., "Cross-talk between dopachrome tautomerase and caveolin-1 is melanoma cell phenotype-specific and potentially involved in tumor progression", Journal of Biological Chemistry, vol. 291, no. 24, p. 12481-12500, 2016. https://doi.org/10.1074/jbc.m116.714733
[6] W. Chu, B. Pak, M. Bani, M. Kapoor, S. Lu, A. Tamiret al., "Tyrosinase-related protein 2 as a mediator of melanoma specific resistance to cis-diamminedichloroplatinum(ii): therapeutic implications", Oncogene, vol. 19, no. 3, p. 395-402, 2000. https://doi.org/10.1038/sj.onc.1203315
[7] A. Tingaud-Sequeira, E. Mercier, V. Michaud, B. Pinson, I. Gazova, E. Gontieret al., "The dct-/- mouse model to unravel retinogenesis misregulation in patients with albinism",, 2022. https://doi.org/10.1101/2022.05.25.493436
[8] A. Tingaud-Sequeira, E. Mercier, V. Michaud, B. Pinson, I. Gazova, E. Gontieret al., "The dct−/− mouse model to unravel retinogenesis misregulation in patients with albinism", Genes, vol. 13, no. 7, p. 1164, 2022. https://doi.org/10.3390/genes13071164

Customer Reviews and Q&A

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■ Q&A

I have a few more questions for you concerning this order:
1. Can we get the non-tagged protein with same purification rate for catalogue product (>90%) ?
2. If the purification rate is less than 90%, can you continue purification process to achieve >90%?
3. What is the isoelectric point?
4. Regarding as lyophilized process, what components is including in the product? We would like to know whether there is some glycerol, sodium or detergents.
5. What is the smallest packing size.
6. Could you tell us the result of small scale expression before complete main production when we receive the order?

Thanks for your inquiry. Here are answers to your questions.
1. Our quality control standards of the protein is higher than 85% as determined by SDS-PAGE. It is not guaranteed that it can reach 90%.
2. There will be more loss of protein for further purification, and it is more difficult to further increase the purity of this no-tagged protein. Considering these two reasons, we will not continue to purify when it reach to our quality control standards.
3. Predicted theoretical pl of this protein is 5.87.
4. Buffer before lyophilization is 10mM Tris-HCI, 1mM M EDTA, pH 8.0. There is no glycerol, sodium or detergents in the lyophilized protein.
5. Regarding this case, our default packing size is 1mg *50vials if you don't have special request. And we could provide 10ug/50ug/100ug/200ug/500ug1mg as well. Pls let us know if you have special request of package.
6. As we mentioned before, we've been working on this case since we got your further inquiry. We've succeeded in removing the tag and the restult of small scale expression is good. So we can make sure to provide 50mg at once.

1) The concentration of this protein is 0.6725 mg/mL before freeze-drying treatment. Is it upper limit of the concentration? I would like to use the protein at a higher concentration.
2) I performed SDS-PAGE at high concentration, but I could not get sharp band. The result shows rather than a ladder bands, like spreading wide, not sharp. I can obtain a clear band at a lower concentration (< 0.6725 mg/mL). As a matter of my experiment, I would like to analyze the protein by SDS-PAGE at high concentration. Could you please advise me to get sharp band at high concentration?
3) Could you please tell me the stability of proteins in water?
4) Have you investigated the isoelectric point of this protein? Furthermore, Is there a difference between CSB-EP006562MO1e1(No tag) and CSB-EP006562MO1(His tag) in the isoelectric point?
5) Could you please provide me with the information regarding the physical properties (e.g. solubility in water or buffer etc...)?

Thanks for your inquiry. Our replies are as below.
1)The lyophilized powder was quantified before lyophilization (liquid state). The protein was measured by the Bradford method, concentration of the protein is 0.6725 mg/ml.
Then according to the package requirements, protein was put into different vials. Just as shown in the COA, (Lyophilized from 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. The volume before lyophilization is 15 ul / vial, 20vial; 75 ul / vial, 20vial; 145 ul / Vial,48vial;720 ul/vial,68vial;1440 ul/vial,10vial)
For example, 10 ug tube: 0.6725 mg/ml*15 ul=10.08 ug. It is the amount of target protein.
2)Freeze-drying only evaporates the water and the ions are not lost. So the lyophilized powder also contains Tris-HCl, EDTA only. It does not contain glycerin, salt, detergent, or any other substances.
3)However, the different volume of reconstituted solution will lead to the difference of its buffer pH and its ionic strength. Therefore, if you want the same ionic concentration and pH before and after lyophilization, you could use deionized sterile water (same volume as before lyophilization) to redissolve.
PS: (If you want to calculate the mass of Tris-HCl, EDTA in each tube, it can be calculated according to the formula (m=c.v.M) where v: the volume before lyophilization)
If the SDS-PAGE is not clear, consider increasing the time of the sample, or prolong the decolorization time, while optimizing the amount of sample loading.
Our running parameters are: Tris-Glycine gel) Discontinuous SDS-PAGE (reduced) with 5% enrichment gel and 15% separation gel, provided for your reference.

Target Background

Function

Catalyzes the conversion of L-dopachrome into 5,6-dihydroxyindole-2-carboxylic acid (DHICA).

Gene References into Functions

soyasaponin Ag inhibited TRP2 expression in a dosedependent manner. Therefore, the depigmenting effect of soyasaponin Ag may be due to the inhibition of tyrosinase expression or the enhancement of tyrosinase degradation. PMID: 28713957
the slaty mutant melanocytes seem to be more sensitive to ultraviolet rays and oxidative stress than black melanocytes. PMID: 26451690
A novel murine model of vitiligo is established by sequential prime/boost immunizations into the hind footpad and tail dermis with tyrosinase-related protein 2 (TRP2)-180 (SVYDFFVWL) peptide. PMID: 23711243
study indicates that tumor antigen (TRP2) expressed in MCMV induces a strong and long-lasting anti-melanoma effect through an antibody-dependent mechanism PMID: 23811402
Vaccine formulations with synthetic long peptides for treating B16F10 skin melanoma fails to elicit strong CD8 T cell responses to self-differentiation antigens TRP-2 and gp100. PMID: 23203930
level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I-SceI to mediate homologous recombination at the Dct locus PMID: 22761925
Recombinant dopachrome tautomerase was expressed and purified, without its carboxy-terminal transmembrane region. Analysis of dopachrome tautomerase tryptic peptides by MALDI-TOF/TOF determined N-glycosylation as a primary post-translational modification. PMID: 20386969
Dopachrome tautomerase inactivation elevates the level of ROS, increases the numbers of sunburn cells and apoptotic cells, and decreases the amount of eumelanin in the epidermis upon exposure to chronic UVA radiation PMID: 20123016
the TRP-2 polypeptide folds in the endoplasmic reticulum (ER) in the presence of calnexin, until it reaches a dithiothreitol-resistant conformation enabling its ER exit to the Golgi PMID: 12719423
Results describe the generation of a knockout of the dopachrome tautomerase gene in mice with effects restricted to pigment production and coat color. PMID: 15060160
We found that ionophore monensin (Mon) and the quaternary amine chloroquine (CQ) discriminate between the traffic routes of TRP-2 and TRP-1 PMID: 15707965
The enzymatic activity of Dct may play a role in determining whether the eumelanin or pheomelanin pathway is preferred for pigment biosynthesis. PMID: 15960609
These results suggest that the slaty mutation affects both eumelanin and pheomelanin synthesis in developmental stage-specific and skin site-specific manners. PMID: 16584806
Slaty mutation blocks the melanosome maturation at stage III and affects the melanosome morphology (elliptical or spherical) in a developmental stage-specific manner. PMID: 17516460
Eumelanin and pheomelanin may accumulate with difficulty in slaty-mutated melanocytes and be easily released from them during skin development. L-Tyr may stimulate this release. PMID: 17551240
These results suggest that the slaty mutation inhibits the development of elliptical stage IV melanosomes and that L-Tyr restores the development of elliptical stage IV melanosomes PMID: 17867832
Cytotoxicity toward targets loaded with a K(b)-restricted tyrosinase-related protein 2-derived peptide correlated with depigmentation in a mouse model of autoimmune vitiligo. PMID: 18337834
mice lacking DCT displayed normal cardiac development but an increased susceptibility to atrial arrhythmias PMID: 19855129

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Involvement in disease

The slaty mutation in Tyrp2 leads to a decrease of DT activity and a consequent change in the pigmentation of the mice to a dark gray/brown eumelanin. The slaty-2j mutation has a similar phenotype, the slaty-lt (light) mutation has a more severe effect and is semidominant; its phenotype may be a result of the failure of the enzyme to be correctly targeted to its normal location on the inner face of the melanosomal membrane.

Subcellular Location

Melanosome membrane; Single-pass type I membrane protein. Melanosome.

Protein Families

Tyrosinase family

Tissue Specificity

Melanocytes and retinal pigmented epithelium (at protein level).

Database Links

KEGG: mmu:13190

STRING: 10090.ENSMUSP00000022725

UniGene: Mm.19987

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