S-layer protein gene of Lactobacillus brevis: cloning by polymerase chain reaction and determination of the nucleotide sequence
| Autor: | Gabriele Vidgren, K. Lounatmaa, Ilkka Palva, Raimo Pakkanen, Airi Palva |
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| Rok vydání: | 1992 |
| Předmět: |
DNA
Bacterial Molecular Sequence Data Biology Microbiology Polymerase Chain Reaction 03 medical and health sciences Bacterial Proteins HSPA2 Protein A/G Consensus sequence Amino Acid Sequence RNA Messenger Cloning Molecular Codon Microscopy Immunoelectron Molecular Biology Peptide sequence 030304 developmental biology Repetitive Sequences Nucleic Acid chemistry.chemical_classification 0303 health sciences Membrane Glycoproteins Base Sequence 030306 microbiology Oligonucleotide Nucleic acid sequence Membrane Proteins Chromosomes Bacterial Molecular biology Peptide Fragments Amino acid Molecular Weight Lactobacillus chemistry Biochemistry Oligodeoxyribonucleotides Genes Bacterial biology.protein Protein G Bacterial Outer Membrane Proteins Research Article |
| Zdroj: | Journal of bacteriology. 174(22) |
| ISSN: | 0021-9193 |
| Popis: | The surface (S)-layer protein of Lactobacillus brevis was isolated, purified, and characterized. The S-layer protein is the major protein of the cell, with an apparent molecular mass of 46 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunogold electron microscopy with polyclonal antiserum against the isolated 46-kDa protein was used to confirm the surface location of this protein. N-terminal amino acid sequences of the intact 46-kDa protein and its tryptic peptides were determined. The gene of the S-layer protein was amplified from the genome of L. brevis by polymerase chain reaction with oligonucleotides, synthesized according to the N-terminal amino acid sequences, as primers. The polymerase chain reaction fragments containing the entire S-layer gene and its regulatory regions were sequenced. Nucleic acid sequence analysis revealed one open reading frame with a capacity to encode a protein of 48,159 Da. From the regulatory region of the gene, two subsequent promoters and a ribosome binding site, showing typical features of prokaryotic consensus sequences, were found. The coding region contained a characteristic gram-positive-type signal peptide of 30 amino acids. Removal of the signal peptide results in a polypeptide of 435 amino acids, which is in excellent agreement with the size of the S-layer protein determined by SDS-PAGE. The size and the 5' end analyses of the S-layer transcripts confirmed the monocistronic nature of the S-layer operon and the functionality of the two promoters found. |
| Databáze: | OpenAIRE |
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