Nitrogen supplementation ameliorates product quality and quantity during high cell density bioreactor studies of Pichia pastoris: A case study with proteolysis prone streptokinase

Streptokinase is a well-established cost-effective therapeutic molecule for thrombo-embolic complications. In the current study, a tag-free variant of streptokinase with a native N-terminus (N-rSK) was developed using the Pichia expression system. A three-copy clone was screened that secreted 1062 mg/L of N-rSK in the complex medium at shake flask level. The biologically active (67,552.61 IU/mg) N-rSK recovered by anion exchange chro-matography was predicted to contain 15.43% alpha-helices, 26.43% beta-sheets. The fermentation run in a complex me-dium yielded a poor quality product due to excessive N-rSK degradation. Therefore, modified basal salt medium was also employed during fermentation operations to reduce the proteolytic processing of the recombinant product. The concomitant feeding of 1 g/L/h soya flour hydrolysate with methanol during the protein synthesis phase reduced the proteolysis and yielded 2.29 g/L of N-rSK. The fermentation medium was also supplemented with urea during growth and induction phases. The combined feeding approach of nitrogen-rich soya flour hy-drolysate and urea during bioreactor operations showed significant improvement in protein stability and re-sulted in a 4-fold increase in N-rSK concentration to a level of 4.03 g/L over shake flask. Under optimized conditions, the volumetric productivity and specific product yield were 52.33 mg/L/h and 33.24 mg/g DCW, respectively.& nbsp; (c) 2021 Elsevier B.V. All rights reserved.

Automated summary

A tag-free variant of streptokinase with improved production and stability was developed using a modified fermentation process and a combined feeding strategy, resulting in a significant increase in yield and protein stability.