HER2/CD340 Protein, Human (HEK293, His)

The HER2/CD340 protein is a dynamic tyrosine kinase that is essential in the neuregulin receptor complex and regulates microtubule dynamics. Upon activation, it triggers the MEMO1-RHOA-DIAPH1 pathway, inhibits GSK3B and promotes the association of APC and CLASP2 on the cell membrane to achieve microtubule stabilization. HER2/CD340 Protein, Human (HEK293, His) is the recombinant human-derived HER2/CD340 protein, expressed by HEK293 , with C-6*His labeled tag.

HER2/CD340 Protein, a dynamic protein tyrosine kinase, stands as a pivotal component within diverse cell surface receptor complexes, requiring a coreceptor for efficient ligand binding. Crucially, it plays an indispensable role as part of the neuregulin-receptor complex, with GP30 identified as a potential ligand for this receptor. Beyond its receptor functions, HER2/CD340 Protein intricately regulates the outgrowth and stabilization of peripheral microtubules (MTs). Upon activation, the MEMO1-RHOA-DIAPH1 signaling pathway, initiated by ERBB2 activation, orchestrates the phosphorylation and subsequent inhibition of GSK3B at the cell membrane. This strategic inhibition prevents the phosphorylation of APC and CLASP2, facilitating their association with the cell membrane. Notably, membrane-bound APC enables the localization of MACF1 to the cell membrane, a prerequisite for microtubule capture and stabilization. Within the nucleus, HER2/CD340 Protein is actively involved in transcriptional regulation, associating with the 5'-TCAAATTC-3' sequence in the PTGS2/COX-2 promoter to activate transcription. Furthermore, its engagement in the transcription of rRNA genes by RNA Pol I enhances protein synthesis, contributing to overall cell growth. The multifaceted activities of HER2/CD340 Protein underscore its central role in orchestrating diverse cellular processes, ranging from receptor signaling to microtubule dynamics and transcriptional regulation.

1.This product does not contain protein kinase domain.
2.Loaded Anti-Human HER2 mAb-Fc on Protein A Biosensor, can bind Human HER2-His with an affinity constant of 1.36 nM as determined in BLI assay.
3.Measured by its ability to block anti-ErbB2 mediated inhibition of SK-BR-3 human breast cancer cell proliferation. The ED₅₀ this effect is ≤1 μg/mL in the presence of 0.6 µg/mL Trastuzmab, corresponding to a specific activity is ≥1×10³ units/mg.
4.Trastuzumab (HY-P9907) immobilized on CM5 Chip can bind Human HER2 with an affinity constant of 0.622 nM as determined in a SPR assay.
5.Loaded Anvatabart (HY-P990697) on AHC2 biosensor, can bind HER2/CD340 Protein, Human (HEK293, His) with an affinity constant of 1.171E-09 M as determined in BLI assay.

• 
  Trastuzumab (HY-P9907) immobilized on CM5 Chip can bind Human HER2 with an affinity constant of 0.622 nM as determined in a SPR assay.
• 
  Loaded Anvatabart (HY-P990697) on AHC2 biosensor, can bind HER2/CD340 Protein, Human (HEK293, His) with an affinity constant of 1.171E-09 M as determined in BLI assay.

SK-BR-3 Cell Proliferation Blocking Assay
Materials
Cell line used for assay: SK-BR-3 cells
Cell culture medium: McCoy's 5A medium + 10% FBS + 1% P/S
Target protein: HER2/CD340 protein, human (HEK293, His) (HY-P70254)
Penicillin-streptomycin (100×) (HY-K1006)
Cell Counting Kit-8 (HY-K0301)
Procedure
1. Seed cells into a 96-well plate at a density of 4,000 cells per well, with a volume of 100 μL per well (do not add cells to the outer wells of the 96-well plate; add an equal volume of PBS solution instead).
2. After the cells have adhered, remove the medium from the wells and starve the cells for 12 hours in medium containing 1% FBS.
3. First, resuspend the protein in ddH₂O to a concentration of 200 μg/mL. Then, dilute the protein with McCoy’s 5A complete medium containing 1% FBS (and 0.6 μg/mL trastuzumab) to a final concentration of 100 μL per well in the 96-well plate, resulting in final HER2 protein concentrations of 0, 0.0001, 0.001, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 50, and 100 μg/mL, respectively.
4. Incubate the cells in a 37°C, 5% CO₂ incubator for 48 hours.
5. After the incubation period, add 10 μL of CCK-8 solution to each well under light-protected conditions, and incubate in a 37°C, 5% CO₂ incubator for 2 hours.
6. Measure the absorbance (OD) at a wavelength of 450 nm using a microplate reader.
7. Using GraphPad Prism software, plot a curve with OD values on the y-axis and protein concentration on the x-axis, and calculate the ED₅₀ value.

Human

HEK293

C-6*His

P04626-1 (T23-T652)

2064  [NCBI]

Extracellular Domain

TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLT

Approximately 88-120 kDa, band on SDS-PAGE under reducing conditions due to the glycosylation.

• 
  ≥ 95%, as determined by reducing SDS-PAGE.

Lyophilized powder

1.Lyophilized from a 0.22 μm filtered solution of 20 mM PB, 150 mM NaCl, pH 7.4.
2.Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4.
Please refer to the lot-specific COA for specific buffer information.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.