US 12,466,852 B2
Method for extraction and purification of hirudin mutant and use thereof
Jiepeng Chen, Shantou (CN); Lili Duan, Shantou (CN); Hongrui Chen, Shantou (CN); Liusong Hu, Shantou (CN); Yufan Chen, Shantou (CN); Lin Hong, Shantou (CN); Zhikai Xu, Shantou (CN); Honglin Ye, Shantou (CN); Yeyu Ji, Shantou (CN); and Chunli Cai, Shantou (CN)
Assigned to SUNGEN BIOSCIENCE CO., LTD., Shantou (CN)
Appl. No. 17/282,303
Filed by SUNGEN BIOSCIENCE CO., LTD., Guangdong (CN)
PCT Filed Apr. 16, 2019, PCT No. PCT/CN2019/082785
§ 371(c)(1), (2) Date Apr. 1, 2021,
PCT Pub. No. WO2020/210965, PCT Pub. Date Oct. 22, 2020.
Prior Publication US 2021/0317162 A1, Oct. 14, 2021
Int. Cl. C07K 1/14 (2006.01); C07K 14/815 (2006.01)
CPC C07K 1/14 (2013.01) [C07K 14/815 (2013.01)] 14 Claims
OG exemplary drawing
 
1. A method for extracting and purifying hirudin mutant HV2-Lys47 (SEQ ID NO.: 1) from a fermentation broth of Escherichia coli for fermentative production of hirudin mutant HV2-Lys47, comprising the steps of:
treating the fermentation broth of Escherichia coli at an elevated temperature to sterilize the fermentation broth and remove impurity proteins;
treating the sterilized fermentation broth by a ceramic membrane to remove the bacteria;
treating the resultant fermentation broth from the ceramic membrane by an ultrafiltration membrane to remove impurity proteins;
treating the resultant fermentation broth from the ultrafiltration membrane by a nanofiltration membrane to obtain a concentrated solution of crudely separated hirudin mutant HV2-Lys47;
adding a salt into the concentrated solution of crudely separated hirudin mutant HV2-Lys47 as an auxiliary material, followed by spray drying to obtain dry powder;
dissolving the dry powder in water and filtering out impurities;
subjecting the filtrate to molecular sieve column chromatography only once, eluting the same with water, and concentrating the collected solution to obtain a crude hirudin mutant HV2-Lys47, wherein the molecular sieve column chromatography is selected from the group consisting of dextran gel G-25, dextran gel G-50, dextran gel G-75, and dextran gel G-100; and
dissolving the crude hirudin mutant HV2-Lys47 in water, adding an organic solvent thereto to precipitate the hirudin mutant HV2-Lys47, and vacuum drying the same to obtain a pure hirudin mutant HV2-Lys47,
wherein the organic solvent is selected from one or more of ethanol, methanol, acetone, or isopropanol, and the volume of the organic solvent added is 5-9 times volumes of the aqueous solution in which crude hirudin mutant HV2-Lys47 is dissolved.