- Structural analysis of N-/O-glycans assembled on proteins in yeasts
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Eun Jung Thak , Jungho Kim , Dong-Jik Lee , Jeong Yoon Kim , Hyun Ah Kang
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J. Microbiol. 2018;56(1):11-23. Published online January 4, 2018
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DOI: https://doi.org/10.1007/s12275-018-7468-x
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 Abstract
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Protein glycosylation, the most universal and diverse posttranslational
modification, can affect protein secretion, stability,
and immunogenicity. The structures of glycans attached
to proteins are quite diverse among different organisms and
even within yeast species. In yeast, protein glycosylation plays
key roles in the quality control of secretory proteins, and particularly
in maintaining cell wall integrity. Moreover, in pathogenic
yeasts, glycans assembled on cell-surface glycoproteins
can mediate their interactions with host cells. Thus, a
comprehensive understanding of protein glycosylation in various
yeast species and defining glycan structure characteristics
can provide useful information for their biotechnological
and clinical implications. Yeast-specific glycans are a target
for glyco-engineering; implementing human-type glycosylation
pathways in yeast can aid the production of recombinant
glycoproteins with therapeutic potential. The virulenceassociated
glycans of pathogenic yeasts could be exploited
as novel targets for antifungal agents. Nowadays, several glycomics
techniques facilitate the generation of species- and
strain-specific glycome profiles and the delineation of modified
glycan structures in mutant and engineered yeast cells.
Here, we present the protocols employed in our laboratory
to investigate the N- and O-glycan chains released from purified
glycoproteins or cell wall mannoproteins in several
yeast species.
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Citations
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Nidia Maldonado-Carmona, Melissa Vázquez-Hernández, Osiris Jair Patiño Chávez, Stefany Daniela Rodríguez-Luna, Omar Jiménez Rodríguez, Sergio Sanchez, Corina Diana Ceapă
Current Opinion in Pharmacology.2019; 48: 1. CrossRef
- Stable Expression and Secretion of Polyhydroxybutyrate Depolymerase of Paucimonas lemoignei in Escherichia coli
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Se Whan Park , Moon Gyu Chung , Hwa Young Lee , Jeong Yoon Kim , Young Ha Rhee
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J. Microbiol. 2008;46(6):662-669. Published online December 24, 2008
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DOI: https://doi.org/10.1007/s12275-008-0283-z
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Abstract
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An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of PhaZ1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing PhaZ1 and its own N-terminal signal peptide (PrePhaZ1) enabled the secretion of active PhaZ1 into the extracellular medium. However, the PrePhaZ1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-PhaZ1 and INPNCPrePhaZ1 fusion constructs did not affect growth of host cells. INPNC-PhaZ1 was successfully displayed on the cell surface with its fusion form, but did not retain PhaZ1 activity. In the case of INPNC-PrePhaZ1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active PhaZ1 was consequently released into the culture medium. The amount of PhaZ1 derived from E. coli (INPNC-PrePhaZ1) was almost twice as great as that directly expressed from E. coli (PrePhaZ1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22°C but was efficiently secreted into the extracellular medium when cultivated at 37°C.
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Citations
Citations to this article as recorded by - Extracellular recombinant protein production from Escherichia coli
Ye Ni, Rachel Chen
Biotechnology Letters.2009; 31(11): 1661. CrossRef
- Controlled Expression and Secretion of Aspergillus oryzae Alkaline Protease in Aspergillus nidulans
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Eun Ah Kim , Jeong Goo Lee , Mi Kyung Whang , Hee Moon Park , Jeong Yoon Kim , Suhn Kee Chae , Pil Jae Maeng
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J. Microbiol. 2001;39(2):95-101.
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Abstract
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In an effort to develop an efficient expression and secretion system for heterologous proteins in Aspergillus nidulans, the PCR-amplified coding sequence for alkaline protease (AlpA) of A. oryzae was cloned into a fungal expression vector downstream of A. nidulans alcA (alcohol dehydrogenase) promoter to yield pRAAlp. Transformation of A. nidulans with pRAAlp gave stable transformants harboring various copy numbers (3 to 10) of integrated alpA gene, from among which 6 representatives were selected. On a medium containing 0.8% ammonium sulfate that represses the expression of the hosts own protease, the alcA promoter-controlled AlpA expression was strongly induced by threonine but repressed by glucose. The level of AlpA secretion was highest (approximately 666 mU/ml) in transformant ALP6 containing the largest copy number integrated alpA. However, the level of AlpA secretion was not necessarily proportional to the copy numbers of the integrated alpA genes. The N-terminal sequence of the secreted mature AlpA was determined to be Gly-Leu-Thr-Thr-Gln-Lys-Ser and its molecular mass to be approximately 34 kDa, indicating that AlpA is properly processed by the removal of 121 N-terminal amino acids.
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