期刊
MICROBIAL CELL FACTORIES
卷 19, 期 1, 页码 -出版社
BMC
DOI: 10.1186/s12934-020-1280-0
关键词
Pichia pastoris; Endoglycosidase; N-GlcNAc-protein; Glycoprotein; Homogeneous protein production
资金
- Shandong province Key RD Program [2019GSF107048]
- National Natural Science Foundation of China [31500648]
- Open Projects Fund of Shandong Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University [2019CCG08]
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) expression 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.
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