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Combining expression and process engineering for high-quality production of human sialyltransferase in Pichia pastoris.

Authors
  • Luley-Goedl, Christiane1
  • Czabany, Tibor2
  • Longus, Karin2
  • Schmölzer, Katharina1
  • Zitzenbacher, Sabine1
  • Ribitsch, Doris1
  • Schwab, Helmut3
  • Nidetzky, Bernd4
  • 1 Austrian Center of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria. , (Austria)
  • 2 Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, 8010 Graz, Austria. , (Austria)
  • 3 Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria. , (Austria)
  • 4 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: [email protected] , (Austria)
Type
Published Article
Journal
Journal of biotechnology
Publication Date
Oct 10, 2016
Volume
235
Pages
54–60
Identifiers
DOI: 10.1016/j.jbiotec.2016.03.046
PMID: 27018228
Source
Medline
Keywords
License
Unknown

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.

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