title: Combining expression and process engineering for high-quality production of human sialyltransferase in Pichia pastoris.
abstract: The human β-galactoside α2,6-sialyltransferase I, ST6Gal-I has drawn considerable interest for its use as biocatalyst for in-vitro glycoengineering of recombinantly produced therapeutic proteins. By attaching sialic acid onto the terminal galactoses of biantennary protein N-glycans, ST6Gal-I facilitates protein remodeling towards a humanized glycosylation and thus optimized efficacy in pharmacological use. Secreted expression of ST6Gal-I in Pichia pastoris is promising, but proteolysis restricts both the yield and the quality of the enzyme produced. Focusing on an N-terminally truncated (Δ108) variant of ST6Gal-I previously shown to represent a minimally sized, still active form of ST6Gal-I, we show here that protein expression engineering and optimization of bioreactor cultivation of P. pastoris KM71H (pPICZαB) synergized to enhance the maximum enzyme titer about 57-fold to 17units/L. N-Terminal fusion to the Flag-tag plus deletion of a potential proteolytic site (Lys(114)-Asn→Gln(114)-Asn) improved the intrinsic resistance of Δ108ST6Gal-I to degradation in P. pastoris culture. A mixed glycerol/methanol feeding protocol for P. pastoris growth and induction was key for enzyme production in high yield and quality. The sialyltransferase was recovered from the bioreactor culture in a yield of 70% using a single step of anion-exchange chromatography. Its specific activity was 0.05units/mg protein.
authors.affiliation: Austrian Center of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Christiane
authors.initials: C
authors.lastname: Luley-Goedl
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, 8010 Graz, Austria.
authors.forename: Tibor
authors.initials: T
authors.lastname: Czabany
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, 8010 Graz, Austria.
authors.forename: Karin
authors.initials: K
authors.lastname: Longus
authors.affiliation: Austrian Center of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Schmölzer
authors.affiliation: Austrian Center of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Sabine
authors.initials: S
authors.lastname: Zitzenbacher
authors.affiliation: Austrian Center of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Doris
authors.initials: D
authors.lastname: Ribitsch
authors.affiliation: Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria.
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
authors.affiliation: Austrian Center of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria; Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, 8010 Graz, Austria. Electronic address: bernd.nidetzky@tugraz.at.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D019149:Bioreactors; D006031:Glycosylation; D006801:Humans; D019158:N-Acetylneuraminic Acid / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012799:Sialyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid
keywords: Expression and process engineering; Glycoengineering; N-Glycosylation; Pichia pastoris; Sialic acid; Sialyltransferase