The Generation and Characterization of Recombinant Protein and Antibodies of Clostridium perfringens Beta2 Toxin

Figure 1

Purification and characterization of recombinant toxin beta2 of Clostridium perfringens type C (rCPB2). The expression of CPB2 was induced by IPTG in E. coli BL21 (DE3) cells harboring His-tagged CPB2 expressing plasmid pTIG-Trx-CPB2; the bacterial cells expressed rCPB2 lysed by sonication, and the soluble fraction of cells was used for purification of rCPB2 by HPLC AKTA PURIFIER PH/C10 system using a Ni-NTA column with a linear gradient elution of 0–1000 mM imidazole. (a) The map of pTIG-Trx-CPB2 plasmid capable of expressing rCPB2 in E. coli cells. (b) An image of the expression and purification of recombinant CPB by a Coomassie stained SDS-PAGE (12%) analysis. Lane M, a ladder of molecular weights (MW). Lane 1, a lysate of E. coli BL21(DE3) cells transformed with plasmid pTIG-Trx. Lane 2, a lysate of E. coli BL21(DE3) cells transformed with plasmid pTIG-Trx-CPB2 induced by IPTG. Lane 3, a fraction of HPLC elution at 10 mM of imidazole. Lane 4, a fraction of HPLC elution at 25 mM of imidazole. Lanes 5–7, fractions of HPLC elution at 75 mM of imidazole. Lane 8, cell lysate of C59-44. Lane 9, the culture supernatant of C59-44 (type C). A band of protein of interest with expected MW of ~28 kDa was observed in lanes 2 and 5–7 (arrowhead). (c) An immunoblotting analysis of the specificity and integrity of purified rCPB2 using monoclonal antibody against CPB2; a specific band was detected in purified fractions of rCPB2 (lanes 5–7); the lysate of E. coli harboring cpb2 recombinant plasmid (lane 2) and supernatant of C. perfringens C59-44 strain (lane 9).

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