Studies Of The Clone And Expression In Pichia Pastoris Of Human Lactoferrin Gene

Pichia Pastoris is a newly developed yeast expression system in recent years. It combines the growth characteristics and molecular genetic background of prokaryotic organisms together with post-translational modifications of eukaryotes, such as: glycosylation and disulfide bond formation et al. More than 200 different proteins have been successfully expressed in the Pichia Pastoris expression system during the past 20 years.Lactoferrin (Mw:=76~80kD) is an iron-binding glycoprotein of transferrin family found in milk, tear, saliva and other secretions. It is also present in the secondary granules of neutrophils. It was proved that the protein has multifunctional properties in vitro and may be involved in regulation of iron homeostasis, inhibition of bacterical and virious growth, and immunilogical regulation through previous studies. Lactoferrin is becoming a hot point all over the world since it was founded.In this research, the total cellular RNAs were isolated from pregnants human neutrophil cells. The cDNA encoding human lactoferrin was amplified by RT-PCR and cloned into pGEM-T easy vector which was then sequenced. The length of hLF cDNA is 2136 bp, and comparison with 8 other hLF cDNA sequences registered in GenBank shows more than 99% homology in cDNA sequence.The gene of hLF was amplified with Pfu Polymerase using pGEM-T easy/hLF vector as template and the size of PCR products was similar to the expected size by agarose gel electrophoresis, then subcloned hLF gene into the yeast expression vector pPIC9K after restriction endonucleases digestion. It was confirmed that the foreign gene had been inserted into the expression plasmid and the ORF was right. The expression pasmid was electroporated into Pichia Pastoris GS115 after digested by Sal I or Sac I . The His+ transformants was selected on MD plates at 30℃ for several days, and decided the phenotypes of methanol utilization of transformants by comparing the growth conditions on MM and MD plates. It was further confirmed if the hLF gene had been integraded into the yeast genome by PCR. According to the phenotypes of His+ transformants, the positive clones were cultured in BMGY/BMMY, inducing by 1% and 2% methanol. We collectedthe supernatants by centrifugation, precipitated the protein by TCA, and detected the foreign protein by SDS-PAGE, ELISA, Dot blot and Western blot.In conclusion,we successfully cloned hLF cDNA and constructed expression plasmid pPIC9K/hLF, explored the conditions of yeast growth, transformation, screening, induction and detecting of foreign protein. As a result, we obtained secretory human lactoferrin in the supernatant detected by SDS-PAGE, ELISA, Dot blot and Western blot.