A Serine-Type Protease Associated with the Cell Wall of Aquifex pyrophilus, a Hyperthermophilic Bacteria
In-Geol CHOI*a, Sung-Hou KIMb, and Yeon Gyu YUa
a Structural Biology Center, Korea Institute of Science and Technology, 39-1 Hwawolkok- Dong, Songbuk-Ku, Seoul Korea
b Department of Chemistry and Lawrence Berkeley National Laboratory, University of California, Berkeley, California, CA94720, USA
A gene coding a serine type protease has been cloned from Aquifex pyrophilus, known to autotrophic hyperthermophilic bacteria located at deepest branch of phylogenetic tree. The gene has an open reading frame of 619 amino acids which containing triad catalytic residues (Asp, His, Ser) as characteristics of serine protease. Comparison to other serine protease and sequence alignment analysis showed that it has an unusual intervening sequence between the second and third catalytic residues and a possible secretion signal at the N-terminus. In contrast other serine proteases, it contains 7 cysteine residues. A partial sequence of the cloned gene was expressed and a polyclonal antibodies were raised. The antibodies specifically react with a 41 kDa cell wall associated protein. When protease activity in the cell wall fraction of Aquifex pyrophilus was measured in SDS-PAGE containing gelatin as substrate, a couple of protease band including a 41 kDa protease were detected. These results indicated that the cloned gene represent a cell wall associated serine type protease. When the cloned gene has been expressed in a yeast expression system, a protease of 43 kDa size which cross-react with the antibodies were observed in the growth medium.
