Gene cloning and heterologous synthesis of a haloalkaliphilic extracellular protease of Natrialba magadii (Nep).
The gene encoding the protease Nep secreted by the haloalkaliphilic archaeon Natrialba magadii was cloned and sequenced. Upstream of the nep gene, a region related to haloarchaeal TATA-box and BRE-like consensus sequences was identified. The nep-encoded polypeptide had a molecular mass of 56.4 kDa, a pI of 3.77 and included a 121-amino acid propeptide not present in the mature Nep. A Tat motif (GRRSVL) was also identified at residues 10-15 suggesting it is a substrate of the Tat pathway. The primary sequence of Nep was closely related to serine proteases of the subtilisin family from archaea and bacteria (50-85% similarity). The nep gene was expressed in Escherichia coli and Haloferax volcanii resulting in production of active Nep protease. In contrast to the recombinant E. coli strains in which Nep activity was only detected in cell lysate, high levels of Nep protein and activity were detected in the culture medium of stationary phase recombinant Hfx. volcanii strains. The Hfx. volcanii synthesized protease was active in high salt, high pH and high DMSO. This study provides the first molecular characterization of a halolysin-like protease from alkaliphilic haloarchaea and is the first description of a recombinant system that facilitates high-level secretion of a haloarchaeal protease.
Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales Universidad Nacional de Mar del Plata, Funes 3250 4 degrees Nivel, Mar del Plata 7600, Argentina. decastro@mdp.edu.ar
Rosana E
RE
De Castro
Diego M
DM
Ruiz
María I
MI
Giménez
María X
MX
Silveyra
Roberto A
RA
Paggi
Julie A
JA
Maupin-Furlow
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D019641:DNA, Archaeal / Q000235:genetics / Q000302:isolation & purification; D006216:Halobacteriaceae / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D017931:DNA Primers; D019641:DNA, Archaeal; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases