Recombinant production and affinity purification of the pore forming toxin using hexa-His tag and expression cassette.

OBJECTIVES

A newly-introduced protein toxin from a sea anemone, namely fragaceatoxin C is a protein with molecular weight of 20 kDa and pore-forming capability against cell membranes has recently grasped great attentions for its function. In this study, its coding sequence cloned as a fusion protein with His-tag for simple production and rapid purification.

MATERIALS AND METHODS

After PCR amplification using and -harboring primers, the gene fragment was cloned into . was used for expression of constructed vector and toxin expression was verified by SDS-PAGE. For one-step purification Ni-NTA sepharose affinity chromatography was employed. For examination of purified toxin function, RBC hemolytic test was conducted.

RESULTS

The results showed that the FraC-coding gene was successfully cloned between and restriction sites and purified with affinity chromatography. Densitometric analysis represented the purity of approximately 97%. Hemolytic test indicated the purified FraC had remarkable lytic activity on RBC and almost lysed 50% of cells at the concentration value of 6.25 nM.

CONCLUSION

The results indicated that not only purified toxin preserved its activity during expression and purification processes but also exerted its function at lower concentrations so that even the 0.09 nM displayed hemolytic effect.