New eGFP Mutant with Intact C- and N-Termini and Affinity for Ni<sup>2+</sup>

Abstract

The green fluorescent protein GFP has long been used in research practice as a molecular tool. It is often used as a fusion partner. To create fusion constructs, target molecules are attached to the N- or C-terminus of GFP. On the other hand, the N- or C-termini of GFP required to create fusion constructs are also used to attach affinity tags that is greatly facilitating purification. Simultaneous introduction of affinity tag and GFP to both or the same end of GFP can create steric hindrances both in the process of biosynthetic folding of the construct and in its affinity purification. This work is devoted to the production of GFP with a His-tag introduced into the polypeptide chain. This work resulted in eGFP157_7H protein with an embedded His-tag and free N- and C-termini to create fusion proteins. The added His-tag will allow purification of the construct with GFP by metal-chelated affinity chromatography under native conditions. The resulting eGFP157_7H variant retained the original fluorescent properties completely similar to those of wild-type eGFP.