Characterization, identification, and cloning of the S-layer protein from Cytophaga sp.
We characterized, identified, and cloned a major protein which comprised 16% of the total proteins from Cytophaga sp. cell lysate. After French pressing, the fraction of cell envelope was treated with 0.2% Triton X-100 to remove cell membranes. Subsequent SDS-PAGE analysis of the Triton X-100-insoluble cell wall revealed a protein of 120 kDa with a pI of 5.4, which was identified by gold immunostaining as the surface (S)-layer protein of this soil bacterium. The nucleotide sequence of the cloned S-layer protein gene (slp) encoding this protein consisted of 3144 nucleotides with an ORF for 1047 amino acids, which included a typical 32-amino acid leader peptide sequence. Amino acid sequence alignment revealed 29-48% similarity between this protein and the S-layer proteins from other prokaryotic organisms. The 120-kDa protein from the Cytophaga sp. cell lysate has been characterized as a member of the S-layer proteins, and the slp gene was cloned and expressed in Escherichia coli. E. coli harboring the plasmid containing the 600- or 800-bp DNA fragment upstream of the initiation codon of the slp gene, in the presence of the reporter gene rsda (raw starch digesting amylase), showed amylase activity in starch containing plate. The putative promoter region of slp located 600 bp upstream of the initiation codon might be used for foreign gene expression.
Chiou
Shiow Ying
SY
Department of Food Science and Biotechnology, National Chung-Hsing University, 250 Kuo Kuang Road, Taichung, 402, Taiwan, R.O.C. chiou@sunrise.hk.edu.tw
Kang
Pei Ling
PL
Liao
Tai Way
TW
Jeang
Chii Ling
CL
D003001:Cloning, Molecular; D003589:Cytophaga / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA
D008562:Membrane Glycoproteins; C055764:S-layer proteins