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

Authors
  • Wang, Shengjun1, 2
  • Rong, Yongheng1
  • Wang, Yaoguang1
  • Kong, Decai3
  • Wang, Peng George4
  • Chen, Min1
  • Kong, Yun1
  • 1 Shandong University, Qingdao, China , Qingdao (China)
  • 2 Sun Yat-sen University, Guangzhou, Guangdong, 510006, China , Guangzhou (China)
  • 3 Heze Municipal Hospital, Heze, Shandong, 274000, China , Heze (China)
  • 4 Georgia State University, Atlanta, GA, 30303, USA , Atlanta (United States)
Type
Published Article
Journal
Microbial Cell Factories
Publisher
BioMed Central
Publication Date
Jan 13, 2020
Volume
19
Issue
1
Identifiers
DOI: 10.1186/s12934-020-1280-0
Source
Springer Nature
Keywords
License
Green

Abstract

BackgroundTherapeutic 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.ResultsIn 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 Asn297 was confirmed via Mass Spectrometry.ConclusionThis 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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