title: Engineering-enhanced protein secretory expression in yeast with application to insulin.
abstract: Adaptation to efficient heterologous expression is a prerequisite for recombinant proteins to fulfill their clinical and biotechnological potential. We describe a rational strategy to optimize the secretion efficiency in yeast of an insulin precursor by structure-based engineering of the folding stability. The yield of a fast-acting insulin analogue (Asp(B28)) expressed in yeast was enhanced 5-fold by engineering a specific interaction between an aromatic amino acid in the connecting peptide and a phenol binding site in the hydrophobic core of the molecule. This insulin precursor is characterized by significantly enhanced folding stability. The improved folding properties enhanced the secretion efficiency of the insulin precursor from 10 to 50%. The precursor remains fully in vitro convertible to mature fast-acting insulin.
authors.affiliation: Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark. thk@novonordisk.com
authors.forename: Thomas
authors.initials: T
authors.lastname: Kjeldsen
authors.forename: Svend
authors.initials: S
authors.lastname: Ludvigsen
authors.forename: Ivan
authors.initials: I
authors.lastname: Diers
authors.forename: Per
authors.initials: P
authors.lastname: Balschmidt
authors.forename: Anders R
authors.initials: AR
authors.lastname: Sorensen
authors.forename: Niels C
authors.initials: NC
authors.lastname: Kaarsholm
mesh_terms: D007328:Insulin / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D007328:Insulin