AMCase/CHIA Protein, Human (HEK293, His)

AMCase/CHIA protein is an enzyme that degrades chitin and chitotriose and is involved in defense against nematodes, fungi, and pathogens.It plays a role in Th2 immune responses, in response to IL-13, and in the induction of inflammation.AMCase/CHIA Protein, Human (HEK293, His) is the recombinant human-derived AMCase/CHIA protein, expressed by HEK293 , with C-His labeled tag.

The AMCase/CHIA protein functions as an enzyme that degrades chitin and chitotriose, suggesting its involvement in defense mechanisms against nematodes, fungi, and other pathogens. Additionally, AMCase/CHIA plays a role in T-helper cell type 2 (Th2) immune response and contributes to the response to IL-13, as well as inflammation induced by IL-13. It stimulates the production of chemokines by pulmonary epithelial cells, showcasing its influence on immune and inflammatory responses. Moreover, AMCase/CHIA is implicated in the protection of lung epithelial cells against apoptosis, promoting the phosphorylation of AKT1. Notably, the protein's function in the inflammatory response and cell protection against apoptosis is inhibited by allosamidin, suggesting a dependency on carbohydrate binding for its proper functioning. The diverse roles of AMCase/CHIA underscore its significance in immune defense, inflammation, and cellular homeostasis.

Measured by its ability to cleave 400 μM 4-Methylumbelliferyl-beta -D-N,N’,N”-triacetylchitotriose (4-MU-TACT). The specific activity is 371.38 pmol/min/µg.

Materials
Assay buffer: 0.1 M MES, pH 5.0
AMCase/CHIA Protein, Human (HEK293, His) (HY-P77869)
Substrate: 4-methylumbelliferyl-β-D-N,N',N'-triacetylchitotrioside, prepared as a 5 mM stock solution in DMSO

Procedure
1. Dilute Human CHIA to 4 ng/μL in assay buffer.
2. Dilute the substrate to 400 μM in assay buffer.
3. Add 50 μL of 4 ng/μL Human CHIA to a black 96-well plate, followed by 50 μL of 400 μM substrate to initiate the reaction. Include a substrate blank with 50 μL of assay buffer and 50 μL of 400 μM substrate. Perform three replicates for both the experimental and control groups.
4. Read the fluorescence in kinetic mode for 5 minutes at excitation and emission wavelengths of 365 nm and 445 nm, respectively, to obtain RFU values.
5. Standard Preparation: Prepare standard dilutions in assay buffer with concentrations of 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM. Perform three replicates for each concentration.
6. Calculate enzyme activity:

Human

HEK293

C-8*His

Q9BZP6 (Y22-A476)

27159  [NCBI]

Full Length of Mature Protein

YQLTCYFTNWAQYRPGLGRFMPDNIDPCLCTHLIYAFAGRQNNEITTIEWNDVTLYQAFNGLKNKNSQLKTLLAIGGWNFGTAPFTAMVSTPENRQTFITSVIKFLRQYEFDGLDFDWEYPGSRGSPPQDKHLFTVLVQEMREAFEQEAKQINKPRLMVTAAVAAGISNIQSGYEIPQLSQYLDYIHVMTYDLHGSWEGYTGENSPLYKYPTDTGSNAYLNVDYVMNYWKDNGAPAEKLIVGFPTYGHNFILSNPSNTGIGAPTSGAGPAGPYAKESGIWAYYEICTFLKNGATQGWDAPQEVPYAYQGNVWVGYDNIKSFDIKAQWLKHNKFGGAMVWAIDLDDFTGTFCNQGKFPLISTLKKALGLQSASCTAPAQPIEPITAAPSGSGNGSGSSSSGGSSGGSGFCAVRANGLYPVANNRNAFWHCVNGVTYQQNCQAGLVFDTSCDCCNWA

55-60 kDa

≥ 95%, as determined by Bis-Tris PAGE.

Lyophilized powder

Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4, 8% trehalose.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O.

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.