DT3C (Diphtheria toxin & spg 3C domain) Active Protein | DT3C active protein

Recombinant DT3C (Diphtheria toxin & spg 3C domain) for Antibody Internalization Assay

Cat. Num. AAA278949

• Purity: Greater than 90% as determined by SDS-PAGE. Greater than 90% as determined by SEC-HPLC.
• Synonyms: DT3C (Diphtheria toxin & spg 3C domain); N/A; Recombinant DT3C (Diphtheria toxin & spg 3C domain) for Antibody Internalization Assay; DT3C active protein

• Host: E Coli
• Purity/Purification: Greater than 90% as determined by SDS-PAGE. Greater than 90% as determined by SEC-HPLC.
• Form/Format: Lyophilized or liquid (Format to be determined during the manufacturing process)
• Sequence Positions: 33-417aa (P00588) & 291-497aa (P19909)
• Sequence: GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKGGGGSGGGGSGGGGSIDEILAALPKTDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE

• Species: Corynephage beta
• Protein Length: Linked Heterodimer
• Tag Info: N-terminal 6xHis-tagged
• Buffer: If the delivery form is liquid, the default storage buffer is Tris/PBS-based buffer, 5%-50% glycerol.If the delivery form is lyophilized powder, the buffer before lyophilization is Tris/PBS-based buffer, 6% Trehalose, pH 8.0.
• 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?/-80?. Our default final concentration of glycerol is 50%. Customers could use it as reference.
• Preparation and Storage: Store at -20 degrees C. For long-term storage, store at -20 degrees C or -80 degrees C. Store working aliquots at 4 degrees C for up to one week. Repeated freezing and thawing is not recommended.

Application Data

(The purity of DT3C was greater than 90% as determined by SEC-HPLC.)

SDS-PAGE

((Tris-Glycine gel) Discontinuous SDS-PAGE (reduced) with 5% enrichment gel and 15% separation gel.)

Related Product Information for DT3C active protein

The recombinant DT3C protein was produced by expressing a DNA fragment encoding the catalytic and translocation domains of the Corynephage beta diphtheria toxin (DT) (33-417aa) fused with the Streptococcal protein G-derived 3C domain (291-497aa) in E.coli. The 6xHis-tag sequence was placed in the N-terminus of the DT3C encoding sequence. The resulting recombinant DT3C protein consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C) [1]. The purity of the recombinant DT3C protein was assessed using SDS-PAGE, demonstrating a purity level of up to 90%. On the gel, DT3C migrated as a band with an approximate molecular weight of 69.4 kDa.

The rationale behind designing the recombinant DT3C protein stems from the cytotoxic property of diphtheria toxin, which inhibits the protein translation machinery in cells, and the Fc-binding domain (3C), capable of binding to any IgG antibodies across different species [1][2]. It binds to a mAb to form a mAb-DT3C conjugate after incubation at room temperature, which functions in vitro similarly to an antibody-drug conjugate (ADC), delivering the cytotoxic effects of diphtheria toxin specifically to targeted cells. Moreover, the mAb-DT3C conjugate serves as a tool to assess the efficiency of mAb internalization by cells by monitoring the extent to which it induces cell death. Additionally, the recombinant DT3C protein facilitates the evaluation of cancer cells' internalization efficiency of the mAb in vitro, aiding in the screening process for more effective monoclonal antibodies.

References

[1] Hamamichi, S., Fukuhara, T., et al. Novel method for screening functional antibody with comprehensive analysis of its immunoliposome [J]. Sci Rep 11, 4625 (2021).
[2] Miki Yamaguchi, Yukari Nishii, et al. Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells [J]. Biochemical and Biophysical Research Communications, Volume 454, Issue 4, 28 November 2014, Pages 600-603.

NCBI and Uniprot Product Information

• NCBI GI #: 118949
• UniProt Accession #: P00588; P19909
• Molecular Weight: 69.4 kDa