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Intracellular proteolysis is attracting more and more attention for its unique and important character in Mycobacterium tuberculosis (Mt).The ClpS protein from Mt (MtClpS) plays a critical role in intracellular proteolysis by recognizing N-end rule substrates,which makes it become a potential target for antibacterial drugs.However,the molecular mechanism of MtCIpS recognizing N-end rule substrates remains unclear.Preparation of highly concentrated and pure MtClpS protein is a prerequisite for further structural and functional studies.In the present work,we tried several fusion tags and various expression conditions to maximize the production of MtClpS in Escherichia coil.We established an efficient approach for preparing the MtClpS protein with a high yield of 24.7 mg/l and a high purity of 98％.After buffer screening,we obtained a stable MtClpS protein sample concentrated at 0.63 mM in the presence of glycerol,I-Arginine,and I-Glutamate.Moreover,circular dichroism characterization indicated that the secondary structure of MtClpS consists of 38％ α-helix and 24％ β-sheet.The 2D 1H-15N HSQC nuclear magnetic resonance spectrum showed a good dispersion of resonance peaks with uniform intensity,indicating that the purified MtClpS protein was well folded and conformationally homogeneous.Isothermal titration calorimetry experiments revealed significant interactions of MtClpS with N-end rule peptides beginning with Leu,Tyr,Trp,or Phe.Furthermore,residues D34,D35,and H66 were confirmed as key residues for MtClpS recognizing the N-end rule peptide.The successful expression and biophysical characterization of MtClpS enabled us to gain insight into the molecular mechanism of MtClpS recognizing N-end rule substrates.The obtained stable and pure recombinant MtClpS will enable future inhibitor screening experiments.