Title
Structural and functional analysis of the S-layer protein crystallisation domain of Lactobacillus acidophilus ATCC 4356 : evidence for protein : protein interaction of two subdomains
Author
Smit, E.
Jager, D.
Martinez, B.
Tielen, F.J.
Pouwels, P.H.
Centraal Instituut voor Voedingsonderzoek TNO
Publication year
2002
Abstract
The structure of the crystallisation domain, SAN, of the S A-protein of Lactobacillus acidophilus ATCC 4356 was analysed by insertion and deletion mutagenesis, and by proteolytic treatment. Mutant S A-protein synthesised in Escherichia coli with 7-13 amino acid insertions near the N terminus or within regions of sequence variation in SAN (amino acid position 7, 45, 114, 125, 193), or in the cell wall-binding domain (position 345) could form crystalline sheets, whereas insertions in conserved regions or in regions with predicted secondary structure elements (positions 30, 67, 88 and 156) destroyed this capacity. FACscan analysis of L. acidophilus synthesising three crystallising and one non-crystallising SA-protein c-myc (19 amino acid residues) insertion mutant was performed with c-myc antibodies. Fluorescence was most pronounced for insertions at positions 125 and 156, less for position 45 and severely reduced for position 7. By cytometric flow sorting a transformant harbouring the mutant SA-protein gene (position 125) was isolated that showed an increased fluorescense signal. Immunofluorescence microscopy suggested that the transformant synthesized mutant SA-protein only. PCR analysis of the transformant grown in the absence of selection pressure indicated that the mutant allele was stably integrated in the chromosome. Proteolytic treatment of SA-protein indicated that only sites near the middle of SAN are susceptible, although potential cleavage sites are present through the entire molecule. Expression in E. coli of DNA sequences encoding the two halves of SAN yielded peptides that could oligomerize. Our results indicate that SAN consists of a ∼12 kDa N and a ∼ 18 kDa C-terminal subdomain linked by a surface exposed loop. The capacity of SA-protein of L. acidophilus to present epitopes, up to ∼ 19 amino acid residues in length, at the bacterial surface in a genetically stable form, makes the system, in principle, suitable for application as an oral delivery vehicle. © 2002 Elsevier Science Ltd. All rights reserved. Chemicals/CAS: amino acid, 65072-01-7; DNA, 9007-49-2; Bacterial Proteins; Epitopes; Membrane Glycoproteins; Membrane Proteins; Peptide Fragments; Proto-Oncogene Proteins c-myc; Recombinant Proteins; surface array protein, bacteria; surface layer protein A, Bacteria; Vaccines
Subject
Biology
Bacteria (microorganisms)
Escherichia coli
Lactobacillus
Lactobacillus acidophilus
Alleles
Amino Acid Sequence
Bacterial Proteins
Binding Sites
Chromosomes, Bacterial
Crystallization
Epitopes
Escherichia coli
Genes, myc
Genetic Vectors
Lactobacillus acidophilus
Lactobacillus casei
Membrane Glycoproteins
Membrane Proteins
Molecular Sequence Data
Molecular Weight
Mutagenesis, Site-Directed
Peptide Fragments
Protein Binding
Protein Structure, Secondary
Protein Structure, Tertiary
Proto-Oncogene Proteins c-myc
Recombinant Proteins
Recombination, Genetic
Transformation, Bacterial
Vaccines
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http://resolver.tudelft.nl/uuid:ef4a3877-7d11-442b-b56d-8e79c7435722
DOI
https://doi.org/10.1016/s0022-2836(02)01135-x
TNO identifier
57665
ISSN
0022-2836
Source
Journal of Molecular Biology, 324 (5), 953-964
Document type
article