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

ABSTRACT:

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 NcoI and HindIII-harboring primers, the gene fragment was cloned into pET-28a(+). Escherichia coli BL21 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

SUBMITTER: Imani M 

PROVIDER: S-EPMC5425919 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature