Biomedical Research 2012 Volume 23 Issue 2 183 Biomedical Research 2012; 23 (2): 183-190 Large-Scale Production of Highly Active Recombinant Omi from Escherichia coli Expressed Soluble Protein Aggregates Md. Mashiar Rahman 1 *, Shahina Akhter 1 *, Dipendra Raj Pandeya 1,2 , Roshan D’Souza 1 , Seong- Tshool Hong 1 1 Department of Microbiology and Genetics and Institute for Medical Science, Chonbuk National University Medical School, Jeonju 561-712, South Korea. 2 Department of Biochemistry, Nepalese Army Institute of Health Sciences, Sanobharayang, Kathmandu, Nepal *These authors contributed equally to this work. Abstract Recent increasing evidences have linked Omi to neuroprotection and the cellular protein qual- ity control system. However, it is very difficult to produce functionally active Omi to study the role of Omi in vitro. Here, we report a method for mass production of functionally active high quality Omi through heterologous Escherichia coli expression. The heterologously expressed Omi was present mostly in soluble cell fraction rather than insoluble cell fraction as inclusion bodies, but the protein in soluble cell fraction had very poor activity. In this method, low con- centration of denaturing agents was added to the lysate for complete solubilization and mono- dispersion of Omi after lysising Escherichia coli. The solubilized and monodisperesed protein sample was purified by Ni-NTA agarose affinity chromatography at denatured condition. The purified denatured Omi was then refolded by a rapid-dilution in an optimized protein refold- ing buffer that was designed by systematic variation of parameters favoring the refolding, preventing aggregate formation and influencing the stability of the folding intermediates. Around 20 mg of properly folded Omi was obtained from 1 liter of bacterial culture by this method which is the highest production reported so far. The biological activity was confirmed by its ability to degrade -casein. This method is a straightforward and efficient purification procedure to obtain a higher yield of proper biologically active Omi and that Omi can be used for the investigation of Omi’s association with neuroprotection in various neurodegenerative diseases. Keywords: Omi, soluble protein aggregates, denature, solubilization, monodispersion, purification, refolding, activity Accepted February 24 2012 Introduction The HtrA family of serine proteases, whose members have been identified in most eukaryotes and prokaryotes, takes part in cells’ protection against stress conditions including heat shock, oxidative stress, inflammation, ischemia/reperfusion, and cancer [1]. The most exten- sively studied member of this family is the E. coli HtrA/DegP that acts as a serine protease degrading mis- folded proteins in the periplasm at high temperatures and as a molecular chaperone at low temperatures [2]. To date, four human homologues, named HtrA1-4 of HtrA have been identified. The common feature of the HtrA family of proteins is a highly conserved trypsin-like ser- ine protease domain and one or two PDZ domains [1]. HtrA2 also known as Omi is the only member of the fam- ily of four mammalian HtrA proteases that has a mito- chondrial localization signal in N-terminus and auto cata- lytically processed into mature form to reveal a Reaper- like amino-terminal motif [3]. In vitro studies showed that Omi is an apoptosis-inducing mitochondrial protein [4, 5]. The neuroprotective role of Omi has been initially identi- fied by the demonstration of neurodegeneration in mice lacking HtrA2 expression or function [6, 7]. Like Alz- heimer's disease-associated proteins, presenilin-1 and oli- gomeric amyloid- [8, 9], fibrilized -synuclein and oli- gomeric prion are thought to be associated with Omi. Therefore, the recombinant production of high quality Omi in substantial quantity is important for its biological and structural studies. E. coli are regarded as an outstanding factory for recom- binant expression of proteins. Heterologously expressed recombinant proteins in E. coli result in either soluble cell