Cloning, Expression and Characterization of Membrane Bound FtsH Protease of Geobacillus kaustophilus
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loning, Expression and Characterization of Membrane Bound FtsH Protease of Geobacillus kaustophilus A. Tüleka, *, F. I. Özdemira, **, and S. S. Ramadhana, *** a
Gebze Technical University, Molecular Biology and Genetics Department, Gebze, 41400 Kocaeli/Turkey *e-mail: [email protected] **e-mail: [email protected] ***e-mail: [email protected] Received March 12, 2020; revised May 13, 2020; accepted July 2, 2020
Abstract—FtsH is a unique enzyme as a membrane-bound ATP-dependent zinc-metalloprotease that degrades integral membrane proteins as well as cytoplasmic proteins. It spans the cell membrane twice and has a large cytoplasmic C-terminal with protease and ATP-binding domain. The ATPase and proteolytic activity of soluble FtsH protease from thermophilic Geobacillus kaustophilus strain DSM 7263T which lacked the transmembrane helices was studied. The 1530 bp gene was cloned in pET14b vector and expressed in Escherichia coli BL21(DE3) cells. The recombinant protein containing 6xHis-Tag at N-terminus was purified and enzyme characterization was performed. The molecular mass of N-terminal truncated FtsH was determined as ~58 kDa from SDS-PAGE and Western blotting analysis. The optimum ATPase activity was observed at 55°C. In addition to ATP, the enzyme is also capable of hydrolyzing CTP and at a lesser extent GTP. The ATPase activity of FtsH was inhibited by 10 mM EDTA and O-phenanthroline, at 65 and 48%, respectively and almost completely inhibited in the presence of 1 mM lactacystin or PMSF. Purified FtsH degraded α-casein efficiently (up to 96–98%) in the presence of 4 mM ATP. Our results showed that without N-terminal transmembrane domain FtsH showed an efficient ATPase and protease activity. Keywords: ATP-dependent protease, FtsH protease, membrane-bound protease, thermophilic, Geobacillus kaustophilus DOI: 10.1134/S0003683820060186
ATP-dependent proteases belong to AAA protein family group, which are associated with various activities in the cell and belong to the NTPase superfamily. In both prokaryotes and eukaryotes, regulated proteolysis is carried out by ATP-dependent proteases such as filamentation temperature-sensitive protein H (FtsH), caseinolytic proteases ClpAP, ClpXP in Escherichia coli and heat shock locus HslU ATPase and HsV peptidase HslUV [1]. FtsH (EC 3.4.24.B20), is unique enzyme as a membrane-bound ATP-dependent zincmetalloprotease. FtsH is ubiquitously found in bacteria, mitochondria and plant chloroplasts [2]. The enzyme degrades not only integral membrane proteins but also cytoplasmic proteins [3, 4]. It plays pivotal roles in the quality control of membrane proteins by rapidly eliminating abnormal ones in prokaryotic organisms as well as in the mitochondria and the chloroplasts of eukaryotic cells. In addition, FtsH protease degrades some short-lived proteins in the cytosol and regulates proteolysis of intact proteins under specific conditions. As proteolysis is energy-demanding and irreversible, degradation of natively folded and active proteins is strictly regulated [5]. Acce
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