Recombinant expression, biophysical and functional characterization of ClpS from Mycobacterium tuberculosis.

Recombinant expression, biophysical and functional characterization of ClpS from Mycobacterium tuberculosis. Acta Biochim Biophys Sin (Shanghai). 2019 Oct 25;: Authors: Guo C, Xiao Y, Bi F, Lin W, Wang H, Yao H, Lin D Abstract 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 MtClpS 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 coli. 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, l-Arginine, and l-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, ind...
Source: Acta Biochimica et Biophysica Sinica - Category: Biochemistry Authors: Tags: Acta Biochim Biophys Sin (Shanghai) Source Type: research