Engineering Saccharomyces Cerevisiae for the Production and Secretion of Affibody Molecules.

Abstract

BackgroundAffibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to address if improvements in terms of yield, ease of production and if purification advantages can be identified. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. Results We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, showed to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind human HER3 as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. ConclusionThis study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching high yields and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.

Improving Therapeutic Protein Secretion in the Probiotic YeastSaccharomyces boulardiiusing a Multifactorial Engineering Approach

“…Both are mainly present in the vacuole, but Prb1p is also present extracellularly. Deletion of these 3 proteases alone or in combination improved secretion of a human serum albumin (HSA) - human parathyroid hormone fusion protein in Pp and three cancer-targeting affibodies in Sc (Gast et al, 2022; M. Wu et al, 2013 ). In Sb , deletion of YPS1, PRB1 and PEP4 improved secretory NPA concentration by 124%, 402% and 390%, respectively ( Figure 5 ).…”