Proteases play pivotal roles in the cellular homeostasis and the recruitment of specific cellular proteins in other pathogens (Butler and others 2006). with MTB. There are about 2 million deaths of active TB annually. Effective novel therapeutics are urgently needed to tackle the inexorable increase of multi- and extensively drug-resistant strains, together with HIV coinfections. The serine proteases from several bacteria and viruses were well-established factors involved in the invasion of mammalian cells, including macrophages (Ohol and others 2010; Muttucumaru and others 2011). MTB serine proteases, such as MycP1 and Rv3610c, are well-documented virulence factors (Zhao and Xie 2011). RIP metalloprotease Rv2869c (Rip1) is an MTB virulence determinant by altering cell envelope composition (Makinoshima and Glickman 2005). PepA (Rv0125), a putative secreted serine protease, selectively stimulates peripheral blood mononuclear cells to proliferate and secrete gamma interferon (Skeiky and others 1999). Proteases play pivotal roles in the cellular homeostasis and the recruitment of specific cellular proteins in other pathogens (Butler and others 2006). can use its protease to degrade the host proteins for nitrogen source (Armstrong 2006). Pathogen proteases contribute to virulence by cleaving host proteins, including immunoglobulins, complement components, and the extracellular matrix proteins (Hu and others 2010). IgA1 protease cleaves human IgA1 to evade the host immune attack (Fernaays and others 2006). secretes three proteases (CPAF, Tsp, cHtrA) into host cells in order to manipulate host signaling and complete the intracellular growth cycle (Zhong 2011). As a predicted serine protease, no report exists concerning the role of Rv3668c in the pathogenChost interaction. As an exceedingly successful typical intracellular pathogen, MTB can maneuver its secreted molecules to maximize the survival odds (Malen and others 2007). Several lines of evidence suggested an important role for MTB Rv3668c: a predicted signal sequence in Rv3668c and the presence in the MTB H37Rv culture filtrates, highly conserved among MTB, paratuberculosis (Cerda-Maira and Darwin 2009), orthologs of DegS, and the S1P of the SigE proteolytic cascade of (Ohol and others 2010). Rv3668c is inducible and potentially involved in persistence. Browsing the Rv3668c expression profiling under various treatments in Gene Expression Omnibus found that capreomycin, a peptide antibiotic for multi-drug resistance TB, can suppress its expression about 1.5-fold (Johansen and others 2006). CDC42EP1 The upregulation of Rv3668c in spp., including model in our recombinant challenge. Materials and Methods Bioinformatics analysis Rv3668c protein sequence feature was found using the proteinCprotein BLAST (BLASTP) program comparison of predicted proteins from MTB H37Rv and other mycobacteria. Eighteen orthologs of Rv3668c protein sequences were downloaded from the NCBI website, including AF2122/97 (Sequence ID: NC_002945.3), MTB H37Rv (Sequence ID: ref|”type”:”entrez-protein”,”attrs”:”text”:”NP_218185.1″,”term_id”:”15610804″NP_218185.1|), MTB SUMu005 (Sequence ID: gb|”type”:”entrez-protein”,”attrs”:”text”:”EFP29033.1″,”term_id”:”308340182″EFP29033.1|), MTB T46 (Sequence ID: gb|”type”:”entrez-protein”,”attrs”:”text”:”EFD11259.1″,”term_id”:”289414019″EFD11259.1|), (Sequence ID: NZ_CM000636.3), M (Sequence ID: ref|YP_001853417.1|), Agy99 (Sequence ID: ref|YP_907742.1|), PYR-GCK (Sequence ID: ref|YP_001132649.1|), PYR1 (Sequence ID: ref|YP_956203.1|), MC2 155 (Sequence ID: ref|YP_890400.1|), Delphinidin chloride sp. JLS (Sequence ID: ref|YP_001073452.1|), sp. MCS (Sequence ID: ref|YP_641970.1|), subsp. K-10 (Sequence ID: ref|NP_959340.1|), ATCC 13950 (Sequencen ID: ref|YP_005335953.1|), ATCC BAA-614 (Sequence ID: gb|”type”:”entrez-protein”,”attrs”:”text”:”EFG77477.1″,”term_id”:”295897895″EFG77477.1|), TN (Sequence ID: ref|NP_302490.1|), (Sequence ID: gb|”type”:”entrez-nucleotide”,”attrs”:”text”:”ANAR01000017.1″,”term_id”:”443383939″ANAR01000017.1|), and 104 (Sequence ID: |”type”:”entrez-nucleotide”,”attrs”:”text”:”NC_008595.1″,”term_id”:”118462219″NC_008595.1). Bacterial strains, media, and growth conditions strains were grown in Middlebrook 7H9 broth (Difco) supplemented with 0.5% glycerol and 0.05% Tween 80, or on Middlebrook 7H10 agar supplemented with 0.5% glycerol at 37C. Delphinidin chloride When necessary, the growth media were supplemented with the antibiotic ampicillin (50?g/mL) and kanamycin Delphinidin chloride (25?g/mL for and 50?g/mL for BL21, and purified. In brief, the PCR products were ligated to the pMD19-T simple vector, Delphinidin chloride and then subcloned into pET32a (+) and pGEX-6p-2. Two recombinant strains, BL21-pET32-Rv3668c and BL21-pGEX-6p-2-Rv3668c, were constructed. Fusion protein expression was induced by isopropyl–D-thiogalactoside (IPTG) at a final concentration of 0.1?mM. Cells were sonicated. The supernatant was purified using Ni-NTA Superflow Cartridge (Qiagen) and Glutathione Sepharose 4B (GE), respectively. The purity of the.