Cloning of feather-degrading minor extracellular protease from Bacillus cereus DCUW: dissection of the structural domains.
Bacterial extracellular proteases play an important role in cell survival and cell-cell communication. A high-molecular-mass minor extracellular protease (Vpr) from a feather-degrading bacterium, Bacillus cereus DCUW, has been reported by our laboratory. In the present study, we cloned and expressed Vpr in Escherichia coli. Complete nucleotide sequencing of this gene predicted that the protease is a member of the serine protease family, and smart domain analysis revealed that the protease consists of an N-terminal signal sequence for secretion, a subtilisin_N sequence that is a signature for N-terminal processing, a catalytic S_8 peptidase domain, and finally a long C-terminal protease-associated (PA) region containing nine intrinsically disordered subdomains. Four truncated constructs of the Vpr protease were cloned and expressed in E. coli. We found that the catalytic domain (amino acid residues 172-583) is sufficient for protease activity. Maturation of the Vpr protease needed both N-terminal and C-terminal processing. We have demonstrated that the oligomerization property is associated with the C-terminal protease-associated domain and also shown that the substrate-binding specificity to raw feather resides in this domain.
Ghosh
Abhrajyoti
A
Dr B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, India.|Department of Biochemistry, University of Calcutta, India.
Chakrabarti
Krishanu
K
Department of Biochemistry, University of Calcutta, India.
Chattopadhyay
Dhrubajyoti
D
Dr B. C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, India.|Department of Biochemistry, University of Calcutta, India.
D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005241:Feathers / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary / Q000502:physiology; D012329:RNA, Bacterial / Q000302:isolation & purification; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D021382:Protein Sorting Signals; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases