Production of Recombinant Human Lysosomal Acid Lipase in Schizosaccharomyces pombe:Development of a Fed-Batch Fermentation and Purification Process

摘要

A fed-batch fermentation process has been developed to enable the production of large quantities of recombinant human lysosomal acid lipase(hLAL;EC 3.1.1.13),in Schizosaccharomyces pombe,for preclinical studies as a potential enzyme therapy drug.Recombinant S.pombe,clone ASP397-21,expressed enzymatically active hLAL in the secreted form.A feedback fed-batch system was used to determine the optimal feed rate of a 50% glucose solution used as the carbon source.The feed rate of the glucose solution was calculated by a computer-aided system according to the equation;F=q_(sf)(VX)/S_(in)specific substrate feed rate gram substrate/gram dry cell weight/h;V,volume of culture broth l;X,cell density gram dry cell weight/l;S_(in),concentration of growth limiting substrate in feed solution gram substrate/gram feed solution).At the time of the initial consumption of glucose in the batch-phase culture,the nutrient supply was automatically initiated by means of monitoring the respiratory quotient change.The obtained profile of the feed rate was applied to the feed forward control fermentation.Finally,the cells were grown up to >50 g dry cell weight/l,and the hLAL expression level was approximately 16,000 V/l.Expressed hLAL protein was purified in a two-step process by hydrophobic interaction and anion exchange chromatographies.Purified recombinant hLAL exhibited a 90-150 kDa broad band upon SDS-PAGE with specific activity of about 300 U/mg.After endoglycosidase H treatment,the band converged to 45 kDa,equal to the calculated molecular weight,suggesting that hLAL produced in S.pombe was hyper-glycosylated.N-terminal analysis of de-glycosylated hLAL revealed that the signal sequence of hLAL was correctly processed in S.pombe.