High-quality production of human α-2,6-sialyltransferase in Pichia pastoris requires control over N-terminal truncations by host-inherent protease activities

Microbial Cell Factories

Research

Christiane Luley-Goedl¹  Peter Augustin¹  Sabine Zitzenbacher¹  Tibor Czabany¹  Doris Ribitsch¹  Katharina Schmölzer¹  Bernd Nidetzky²  Helmut Schwab³  Harald Sobek⁴  Rainer Müller⁴  Marco Thomann⁵  Christine Jung⁵ 

[1] ACIB - Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010, Graz, Austria;ACIB - Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010, Graz, Austria;Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010, Graz, Austria;ACIB - Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010, Graz, Austria;Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, A-8010, Graz, Austria;Roche Diagnostics GmbH, Nonnenwald, D-82377, Penzberg, Germany;Roche Diagnostics GmbH, Pharma Biotech Development, Nonnenwald, D-82377, Penzberg, Germany;

关键词: Therapeutic glycoproteins;    In-vitro;    Human sialyltransferase;    ST6Gal-I;    N;    Pichia pastoris;   

DOI  :  10.1186/s12934-014-0138-8

 received in 2014-05-21, accepted in 2014-09-04,  发布年份 2014

来源: Springer

【 摘 要 】

Backgroundα-2,6-sialyltransferase catalyzes the terminal step of complex N-glycan biosynthesis on human glycoproteins, attaching sialic acid to outermost galactosyl residues on otherwise fully assembled branched glycans. This “capping” of N-glycans is critical for therapeutic efficacy of pharmaceutical glycoproteins, making the degree of sialylation an important parameter of glycoprotein quality control. Expression of recombinant glycoproteins in mammalian cells usually delivers heterogeneous N-glycans, with a minor degree of sialylation. In-vitro chemo-enzymatic glycoengineering of the N-glycans provides an elegant solution to increase the degree of sialylation for analytical purposes but also possibly for modification of therapeutic proteins.ResultsHuman α-2,6-sialyltransferase (ST6Gal-I) was secretory expressed in P.pastoris KM71H. ST6Gal-I featuring complete deletion of both the N-terminal cytoplasmic tail and the transmembrane domain, and also partial truncation of the stem region up to residue 108 were expressed N-terminally fused to a His or FLAG-Tag. FLAG-tagged proteins proved much more resistant to proteolysis during production than the corresponding His-tagged proteins. Because volumetric transferase activity measured on small-molecule and native glycoprotein acceptor substrates did not correlate to ST6Gal-I in the supernatant, enzymes were purified and characterized in their action on non-sialylated protein-linked and released N-glycans, and the respective N-terminal sequences were determined by automated Edman degradation. Irrespective of deletion construct used (Δ27, Δ48, Δ62, Δ89), isolated proteins showed N-terminal processing to a highly similar degree, with prominent truncations at residue 108 - 114, whereby only Δ108ST6Gal-I retained activity. FLAG-tagged Δ108ST6Gal-I was therefore produced and obtained with a yield of 4.5 mg protein/L medium. The protein was isolated and shown by MS to be intact. Purified enzyme exhibited useful activity (0.18 U/mg) for sialylation of different substrates.ConclusionsFunctional expression of human ST6Gal-I as secretory protein in P.pastoris necessitates that N-terminal truncations promoted by host-inherent proteases be tightly controlled. N-terminal FLAG-Tag contributes extra stability to the N-terminal region as compared to N-terminal His-Tag. Proteolytic degradation proceeds up to residues 108 – 114 and of the resulting short-form variants, only Δ108ST6Gal-I seems to be active. FLAG-Δ108ST6Gal-I transfers sialic acids to monoclonal antibody substrate with sufficient yields, and because it is stably produced in P.pastoris, it is identified here as an interesting glycoengineering catalyst.

【 授权许可】

CC BY   
© Ribitsch et al.; licensee BioMed Central Ltd. 2014