Legumain 蛋白, Mouse (HEK293, C-His)

Legumain proteins can slowly cleave aspartyl bonds, especially under acidic conditions.Legumain Protein, Mouse (HEK293, C-His) is the recombinant mouse-derived Legumain protein, expressed by HEK293 , with C-10*His labeled tag.

Legumain 蛋白对天冬酰胺键的水解表现出严格的特异性。此外，它还表现出缓慢裂解天冬氨酰键的能力，特别是在酸性条件下，进一步扩展了其酶的多功能性。从功能上讲，Legumain 是溶酶体/内体系统内 MHC II 类抗原呈递蛋白质加工不可或缺的一部分。最近的研究结果表明，它还通过促进 Perforin-2 (MPEG1) 的裂解和成熟，从而促进细胞质中的抗原易位，从而在交叉呈递树突状细胞的 MHC I 类抗原呈递中发挥关键作用。此外，Legumain 对于肾近曲小管中正常溶酶体蛋白降解至关重要，并且是内化 EGFR 降解所必需的，这凸显了其在细胞过程和细胞增殖调节中的重要性。

1. Measured in a cell proliferation assay using RAW264.7 cells. The ED₅₀ this effect is 0.07438ng/mL, corresponding to a specific activity is 1.344×10⁷ units/mg.
2. Measured by its ability to cleave the fluorogenic peptide substrate, N-carbobenzyloxy-Ala-Ala-Asn-7-amido-4-methylcoumarin (Z-AAN-AMC). The specific activity is >130 pmol/min/µg, as measured under the described conditions. (Activation description: The proenzyme needs to be activated in acid buffer for an activated form.)

• 
  Measured in a cell proliferation assay using RAW264.7 cells.The ED₅₀ for this effect is 0.07438 ng/mL, corresponding to a specific activity is 13444474.3211 units/mg.

Materials
Activation buffer: 0.1 M NaOAc, 0.1 M NaCl, pH 4.5
Assay buffer: 50 mM MES, 250 mM NaCl, pH 5.5
Legumain Protein, Mouse (HEK293, C-His) (HY-P70216A)
Substrate: Z-Ala-Ala-Asn-AMC (HY-136626), dissolved in DMSO to 10 mM
Standard: 7-Amino-4-methylcoumarin (AMC, HY-D0027)

Procedure
1. Preparation of Standard Curve: Dilute AMC with assay buffer to concentrations of 10000, 5000, 2500, 1250, 625, 312, 156, 78, 39, 19.5, and 0 pmol respectively. Add 100 μL of each dilution to the wells of a black microplate. Read in kinetic mode for 5 minutes using excitation and emission wavelengths of 380 nm and 460 nm (top read). Generate a standard curve with the measured RFU as the ordinate and the amount of standard substance as the abscissa, and derive the standard curve equation.
2. First, reconstitute the test protein with sterile water to 500 μg/mL, then dilute it to 50 μg/mL with activation buffer, and incubate at 37°C for 4 hours.
3. Dilute the protein solution to 2 ng/μL with assay buffer.
4. Dilute the substrate to 200 μM with assay buffer.
5. Initiate the reaction by adding 50 μL of 2 ng/μL Mouse Legumain and 50 μL of 200 μM substrate to each well. For blank wells, add 50 μL of assay buffer and 50 μL of 200 μM substrate.
6. Read in kinetic mode for 5 minutes using excitation and emission wavelengths of 380 nm and 460 nm.
7. Calculate the specific activity:

Mouse

HEK293

C-His

O89017 (V18-Y435)

19141  [NCBI]

Full Length of Mature Protein

VPVGVDDPEDGGKHWVVIVAGSNGWYNYRHQADACHAYQIIHRNGIPDEQIIVMMYDDIANSEENPTPGVVINRPNGTDVYKGVLKDYTGEDVTPENFLAVLRGDAEAVKGKGSGKVLKSGPRDHVFIYFTDHGATGILVFPNDDLHVKDLNKTIRYMYEHKMYQKMVFYIEACESGSMMNHLPDDINVYATTAANPKESSYACYYDEERGTYLGDWYSVNWMEDSDVEDLTKETLHKQYHLVKSHTNTSHVMQYGNKSISTMKVMQFQGMKHRASSPISLPPVTHLDLTPSPDVPLTILKRKLLRTNDVKESQNLIGQIQQFLDARHVIEKSVHKIVSLLAGFGETAERHLSERTMLTAHDCYQEAVTHFRTHCFNWHSVTYEHALRYLYVLANLCEAPYPIDRIEMAMDKVCLSHY

Approximately 55-60 kDa, based on SDS-PAGE under reducing conditions, due to the glycosylation.

• 
  Greater than 95% as determined by reducing SDS-PAGE.

无菌粉末

Lyophilized from a 0.22 μm filtered solution of 50 mM Tris-HCl/Tris, 300 mM NaCl, pH 7.4.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<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.