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

- The N-terminally truncated variant Δ108 of human sialyltransferase STGal-I was produced in Pichia pastoris.
- Protection of the N-terminal region was important to prevent protein degradation.
- Expression and process engineering were combined to gain 17 units/L of bioreactor culture.
- Mixed glycerol/methanol feed was key for high enzyme titers.
- Enzyme was recovered efficiently using anion exchange chromatography.

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 17 units/L. N-Terminal fusion to the Flag-tag plus deletion of a potential proteolytic site (Lys114-Asn → Gln114-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 0.05 units/mg protein.