PRODUCTION METHOD OF RECOMBINANT HUMAN LACTOFERRIN, N-LOBE LACTOFERRIN, AND C-LOBE LACTOFERRIN USING PICHIA PASTORIS LF2 (KFCC 11106), PICHIA PASTORIS NFL3 (KFCC 11107), AND PICHIA PASTORIS CFL3 (KFCC 11105)

摘要

PURPOSE: A concentration of lactoferrin which is physiologically activating protein, in the milk is low compared with in fore milk, so addition of lactoferrin is required. Lactoferrin having antibiotic activity and regulation of iron uptake in the intestine of farm animals is an important ingredient of animal feed stuff. A production method of lactoferrin using Pichia pastor is provided. CONSTITUTION: cDNAs for lactoferrin, N-lobe lactoferrin, and C-lobe lactoferrin are amplified by PCR. Amplified PCR products are modified by restriction of signal sequence and then ligated with pPIC9K vector. The vector is introduced into E. coli Top10F' strain and transforms it. Each recombinant vector, purified from E. coli is introduced into histidine auxotroph Pichia pastor is and transforms it. Correct colonies are selected. Second round selection is performed on the ground of kanamycin resistance. Using SDS-PAGE and PCR amplification, expression of each protein is confirmed. Preinduction with glycerol and induction with methanol are used to produce each protein. After the main culture, the culture broth and cells are freeze dried and mixed in the animal feed stuff.