High Level Expression And Purification Of Human Serum Albumin In Pichia Pastoris

Human serum albumin(HSA)has been extensively used in a series of clinical care settings for several decades and become the drug protein with maximum usage.To date,HSA has been produced from human blood by cold ethanol precipitation,but the industrial production of this protein is seriously limited by the shortage of human plasma,the diversity of the blood sources and the potential risk for transmission of blood-derived infectious pathogens.After decades of hard work,recombinant HSA(rHSA)has been produced from several expression systems.Among them,Pichia pastoris shows attractive potential for HSA expression by virtue of several advantages and the production has been improved to around 11 g/L.However,the methanol induction time in these studys is so long(over 200 h)that the production efficiency is not impressive and it raises the potential safety hazard derived from storing large volumes of methanol for long time.For the purpose of increasing production efficiency,the hsa gene was cloned and expressed in P.pastoris GS115 in the present work.By screening for histidine autotrophic and a high concentration of geneticin resistance,a recombinant transformant that could effectively express 585 mg/L rHSA after 96 h induction in shake flask was identified.The production was improved to 1.2 g/L by optimization of induction temperature,pH and methanol dosage,and further to 1.6 g/L by addition of 1 g/L alanine,glutamic acid,leucine,lysine,0.5% casanimo acids and 10 mM ascorbic acid.We also can achivied 1.7 g/L rHSA merely by simplifying the BMGY/BMMY medium without any additional supplyments.Thereafter,production profiles of rHSA by the transformant in a 5 L bioreactor were investigated in detail.The result indicated that the classic constant feeding strategy was superior to dissolved oxygen-stat feeding strategy and we achived 8.86 g/L rHSA after 96 h induction [92.29 mg/(L*h)].Finally,a purification procedure which contains affinity chromatography and hydrophobic interaction chromatography was launched rendering a rHSA with over 99.67% purity and around 40% recovery.This study reveals that Pichia pastoris is an excellent system for recombinant human serum albumin expression due to its outstanding expression capacity.To our best knowledge,this is the first report of a high space-time yield of 92.29 mg/(L*h)of rHSA at the bioreactor level where a high broth titer of 8.86 g/L rHSA was also achieved.That,accompanied with the established purification protocol,make it promising for the industrial production of the undersupplied human serum albumin.