Production of a new construct using intein-elastin like fusion protein for simple and non-chromatographic purification of the recombinant protein

Abstract

Intein (INT) or internal protein is a self-cleavage splicing element used for the protein purification and simple bioseparation. INT in fused form to chitin binding domain (CBD) produce an affinity protein purification method for the recombinant proteins. On the other hand elastin-like protein (ELP) is a synthetic biopolymer consisting of the pentapeptide repeats of VPGXG (X is any amino acid except proline). ELP is a thermal responsive polypeptide and contains a reversible transition stage. For each ELP molecule a transition temperature (Tt) is determined so that aggregation can take place after temperature increases while it will solubilize after a decrease of temperature from Tt. The focus of this work is ELP-fused INT sequence design for simple purification of the recombinant protein. In this work ELP₁₀ (150 bp) was synthesized and cloned into pBluescript. Then Elp₆₀ (900 bp) was produced using recursive directional ligation (RDL) method. Finally, ELP₆₀ was subcloned into pTXB1 (an INT-CBD expression vector) and ELP₆₀ expression was confirmed using SDS-PAGE method. Additionally INT-ELP₆₀ fusion proteins were purified using two sequential steps of inverse transition cycle (ITC). The data was shown; ELP₆₀ was expressed and successfully purified in one step and non-chromatographic method. We propose that constructed pTX-INT-ELP can be used for simple and easy purification of the recombinant proteins.

Keywords: Intein, elastin liked protein, expression, Inverse transition cycling, purification.