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Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei
- Source :
- Carbohydrate Research. 345:1422-1431
- Publication Year :
- 2010
- Publisher :
- Elsevier BV, 2010.
-
Abstract
- The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051(T) possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids. The mature S-layer protein has a theoretical molecular mass of 105.95kDa and a calculated pI of 5.83. It contains three S-layer homology domains at the N-terminus that are involved in anchoring of the glycoprotein via a non-classical, pyruvylated secondary cell wall polymer to the peptidoglycan layer of the cell wall. For this polymer, several putative biosynthesis enzymes were identified upstream of the spaA gene. For in vivo cell surface display, the hexahistidine tag and the enhanced green fluorescent protein, respectively, were translationally fused to the C-terminus of SpaA. Immunoblot analysis, immunofluorescence staining, and fluorescence microscopy revealed that the fused epitopes were efficiently expressed and successfully displayed via the S-layer glycoprotein matrix on the surface of P. alvei CCM 2051(T) cells. In contrast, exclusively non-glycosylated chimeric SpaA proteins were displayed, when the S-layer of the glycosylation-deficient wsfP mutant was used as a display matrix.
- Subjects :
- Signal peptide
Glycosylation
Paenibacillus alvei
Recombinant Fusion Proteins
Molecular Sequence Data
ved/biology.organism_classification_rank.species
Protein Engineering
Biochemistry
Article
Analytical Chemistry
Open Reading Frames
chemistry.chemical_compound
Bacterial Proteins
Cell Wall
Amino Acid Sequence
Peptide sequence
Phylogeny
Glycoproteins
chemistry.chemical_classification
Base Sequence
ved/biology
Organic Chemistry
Structural gene
General Medicine
Protein engineering
Molecular biology
Carbohydrate Sequence
chemistry
Genetic Loci
Peptidoglycan
Glycoprotein
Paenibacillus
S-layer
Subjects
Details
- ISSN :
- 00086215
- Volume :
- 345
- Database :
- OpenAIRE
- Journal :
- Carbohydrate Research
- Accession number :
- edsair.doi.dedup.....b2196f5f7a3ea20cf11bf5c118fdca60