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. 2019 Jul 31;4(7):12880–12895. doi: 10.1021/acsomega.9b00399

Figure 2.

Figure 2

Molecular characterization of L. interrogans ClpP1, ClpP2, and ClpX. (A) Cloning and overexpression of ClpPs and ClpX in E. coli. The Coomassie-blue stained 12% SDS-polyacrylamide gel showing the resolved protein lysates of E. coli cells overexpressing LeptospiraclpP1 (pET23a-clpP1), clpP2 (pET23a-clpP2), and clpX (pET23a-clpX), after induction with (+) or without (−) 1 mM IPTG. (B) The affinity-purified Leptospira recombinant proteins. The Ni-NTA affinity-purified rClpP1 (∼23 kDa), rClpP2 (∼22 kDa), and rClpX (∼47 kDa) proteins were resolved on 12% SDS-polyacrylamide gel and stained with Coomassie-blue. In image (A,B), M denotes the standard protein molecular size marker (in kDa). (C,D) Western blot of native ClpP1 and ClpP2 of Leptospira, respectively. The native ClpP1 and ClpP2 in Leptospira lysates were detected in pathogenic strains (serovar Lai and Copenhageni) but not in the nonpathogenic strain (serovar Patoc) using the primary antibodies generated against rClpP1 and rClpP2, respectively. Native and recombinant ClpP isoforms were detected at a similar molecular size.