Homogeneous production and characterization of recombinant N-GlcNAc-protein in Pichia pastoris

Abstract Background Therapeutic glycoproteins have occupied an extremely important position in the market of biopharmaceuticals. N -Glycosylation of protein drugs facilitates them to maintain optimal conformations and affect their structural stabilities, serum half-lives and biological efficiencies. Thus homogeneous N -glycoproteins with defined N -glycans are essential in their application in clinic therapeutics. However, there still remain several obstacles to acquire homogeneous N -glycans, such as the high production costs induced by the universal utilization of mammalian cell expression systems, the non-humanized N -glycan structures and the N -glycosylation microheterogeneities between batches. Results In this study, we constructed a Pichia pastoris ( Komagataella phaffii ) e xpression system producing truncated N -GlcNAc-modified recombinant proteins through introducing an ENGase isoform (Endo-T) which possesses powerful hydrolytic activities towards high-mannose type N -glycans. The results showed that the location of Endo-T in different subcellular fractions, such as Endoplasmic reticulum (ER), Golgi or cell membrane, affected their hydrolytic efficiencies. When the Endo-T was expressed in Golgi, the secreted IgG1-Fc region was efficiently produced with almost completely truncated N -glycans and the N -GlcNAc modification on the glycosite Asn 297 was confirmed via Mass Spectrometry. Conclusion This strategy develops a simple glycoengineered yeast expression system to produce N -GlcNAc modified proteins, which could be further extended to different N -glycan structures. This system would provide a prospective platform for mass production of increasing novel glycoprotein drugs.