Structural and Functional Analysis of the S-layer Protein Crystallisation Domain of Lactobacillus acidophilus ATCC 4356: Evidence for Protein–Protein Interaction of two Subdomains
| Autor: | Peter H. Pouwels, Dennis Jager, Beatriz Martínez, Egbert Smit, Frans J. Tielen |
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| Rok vydání: | 2002 |
| Předmět: |
Genetic Vectors
Molecular Sequence Data Mutant Genes myc Biology medicine.disease_cause Protein Structure Secondary Proto-Oncogene Proteins c-myc Epitopes Protein structure Lactobacillus acidophilus Bacterial Proteins Structural Biology Escherichia coli medicine Amino Acid Sequence Molecular Biology Peptide sequence Alleles Recombination Genetic chemistry.chemical_classification Vaccines Binding Sites Membrane Glycoproteins Membrane Proteins Chromosomes Bacterial Molecular biology Peptide Fragments Recombinant Proteins Protein Structure Tertiary Amino acid Molecular Weight Lacticaseibacillus casei Biochemistry Membrane protein chemistry Mutagenesis Site-Directed Transformation Bacterial Crystallization S-layer Protein Binding |
| Zdroj: | Journal of Molecular Biology. 324:953-964 |
| ISSN: | 0022-2836 |
| Popis: | 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 |
| Databáze: | OpenAIRE |
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