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.
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 S(A)-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 S(A)-protein gene (position 125) was isolated that showed an increased fluorescense signal. Immunofluorescence microscopy suggested that the transformant synthesized mutant S(A)-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 S(A)-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 approximately 12kDa N and a approximately 18kDa C-terminal subdomain linked by a surface exposed loop. The capacity of S(A)-protein of L.acidophilus to present epitopes, up to approximately 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.
authors.affiliation: Department of Applied Microbiology and Gene Technology, TNO Nutrition and Food Research Institute, Utrechtseweg 48, 3700 AJ, Zeist, The Netherlands.
authors.forename: Egbert
authors.initials: E
authors.lastname: Smit
authors.forename: Dennis
authors.initials: D
authors.lastname: Jager
authors.forename: Beatriz
authors.initials: B
authors.lastname: Martinez
authors.forename: Frans J
authors.initials: FJ
authors.lastname: Tielen
authors.forename: Peter H
authors.initials: PH
authors.lastname: Pouwels
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002876:Chromosomes, Bacterial / Q000235:genetics; D003460:Crystallization; D000939:Epitopes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli; D016259:Genes, myc / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007779:Lactobacillus acidophilus / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D007780:Lacticaseibacillus casei / Q000235:genetics; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D014169:Transformation, Bacterial; D014612:Vaccines / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; C055764:S-layer proteins; D014612:Vaccines; C070683:surface layer protein A, Bacteria