A novel feeding strategy for enhanced protein production by fed-batch fermentation in recombinant Pichia pastoris

A novel feeding strategy for enhanced protein production of hepatitis B virus surface antigen (HBsAg) in fed-batch fermentation, recombinant Pichia pastoris, has been developed. A minimal salt medium was used to grow cells in the initial batch fermentation, followed by a glycerol + salts fed-batch phase. At the end of the fed-batch phase a dry cell weight of 130 g l(-1) was achieved. In the absence of basal salts, the same amount of glycerol feed resulted in only 90 g l(-1) cell dry weight. When a limited amount of casamino acids were also included every 24 h during methanol induction, there was a two-fold increase in expression levels of HBsAg. After 192 h of induction, the expression levels of HBsAg (soluble and insoluble) reached > 1 g l(-1) using the Mut(-) strain. Thus, the use of basal salts in the glycerol feed, along with the addition of limited amounts of casamino acids with the methanol feed, resulted in an increased expression of total HBsAg. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.