Study Of Recombinant Human Serum Albumin Production In Pichia Pastoris And Related Process Of Fermentation In Pilot-scale

The major physiological function of Human serum albumin (human serumalbumin, HSA) is to transport and maintain a relatively stable environment includinganti-coagulation, scavenging free radicals, maintaining normal blood osmotic pressure,etc. At present, the resource of HSA in application is extracted from healthy humanplasma. Due to the limited source, the production can barely meet market demand. Asthe rapid development of genetic engineering technology, Japanese Mitsubishi TanabePharma, British DELTA and NCPC companices have been committed in theimprovement of the industrialization process of yeast recombinant expression of HSA.Pichia expression system is widely used, in which the expression of recombinanthuman serum albumin (Recombinant Human Serum Albumin, rHSA)is controlled viamethanol alcohol oxidase promoter (pAOX1) induction. Considering the amount ofrHSA used in clinical treatment, the production of fermentation scale is at least30000L, which indicates large amount of methanol is needed. In addition, it is hard tostore and use methanol, and this severely affects the fermentation safety. To resolvethese problems, this study suggests that glycerol dehydrogenase promotertriphosphate (pGAP) can be used in recombinant Pichiapastoris expression system forrHSA expression. Glycerol is the only carbon source in the fermentation process ofthis system. The production process is simplified and the fermentation duration isshortened. The detailed study is described as follows:This study optimized HSA coding sequence according to the Pichiapastorispreference and transferred into the expression vector, pGAPZaA, and electricitytransformed into the X-33strain. The positive strains were screened using Zeocinresistant medium and PCR. The expression of rHSA in flasks was identified bySDS-PAGE and Western Blotting. The optimal condition of rHSA expression in X–33was studied. The pH value ofmedium was adjusted to4.0,5.0and6.0, respectively, for72h cell culture. Afterwards,the protein expression was measured, and the result indicated the highest productionof rHSA was at pH6.0. The optimal nitrogen source was also studied. Fourfermentation tanks were filled with sterilized water,(NH4)2SO4,(NH4) H2PO4andH3PO4, respectively and cultured for72h. By measuring the protein expression, theaddition of (NH4)H2PO4was found the highest. Hence,(NH4)H2PO4was optimal fornitrogen source addition. The preliminary exploration of Pilot-scale process study onrHSA fermentation provided foundations to rHSA industrial production in type X–33yeast expression system.