Production, purification and characterization of recombinant yeast processing alpha 1,2-mannosidase.
The Saccharomyces cerevisiae processing alpha 1,2-mannosidase, which trims Man9GlcNAc to Man8GlcNAc, has a lumenally oriented catalytic domain and an N-terminal transmembrane domain. To obtain sufficient protein to study the structure and mechanism of action of this enzyme, the sequence encoding the catalytic domain was inserted downstream of the alpha-factor promoter and signal peptide in a high-copy vector for expression in S. cerevisiae as a secreted protein. Using oligosaccharide substrate (Glc1Man9GlcNAc or Man9GlcNAc), the medium of cells transformed with this plasmid showed an increase in alpha-mannosidase activity that was directly related to the increase in cell density, whereas no alpha-mannosidase activity was detected in cells transformed with vector alone. SDS-PAGE of the medium showed the presence of a doublet of 63 and 60 kDa that was revealed by Coomassie Blue staining and by Western blotting with antibodies to the endogenous solubilized alpha-mannosidase. The recombinant alpha-mannosidase was present in the medium at a level of approximately 1 mg/l and was purified in a single step by chromatography on S-Sepharose. High-resolution 1H NMR analysis of the Man8GlcNAc formed from Man9GlcNAc in the presence of the recombinant enzyme proved that it retained its specificity and removed only one specific alpha 1,2-mannose residue of the alpha 1,3 branch. Endoglycosidase H treatment decreased the molecular mass of both components of the doublet by approximately 5 kDa, showing that the heterogeneity is not due to differential N-glycosylation. EDTA inhibited the activity of the recombinant enzyme, but the inhibition was reversed by the addition of divalent cations.(ABSTRACT TRUNCATED AT 250 WORDS)[1]References
- Production, purification and characterization of recombinant yeast processing alpha 1,2-mannosidase. Lipari, F., Herscovics, A. Glycobiology (1994) [Pubmed]
Annotations and hyperlinks in this abstract are from individual authors of WikiGenes or automatically generated by the WikiGenes Data Mining Engine. The abstract is from MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.About WikiGenesOpen Access LicencePrivacy PolicyTerms of Useapsburg
