Isolation and identification of hybrid recombinant E7 oncoprotein of type 16 human papilloma virus conjugated with heat shock protein 70

Abstract

The recombinant E7 oncoprotein gene of type 16 human papilloma virus (conjugated with the heat shock protein 70 (E7(16)-HSP70)) was expressed in Saccharomyces cerevisiae yeast cells. A number of chromatographic isolations were conducted to obtain the target protein and its primary identification and to establish its originality. The use of metal chelate chromatography was based on selective binding of sorbent active groups with a poly-histidine fragment at C-terminus of the target protein polypeptide sequence, while the use of affine chromatography was based on the association with ATP-binding regions of E7 and HSP70. The degree of purity of the target protein after both procedures did not exceed 70%, indicating the presence of many low-molecular admixtures and products of the target molecule oligomerization. The use of chromatographic purification under denaturing conditions using strong reducing agents allowed isolation of the target protein, the monomer content in which exceeded 97%. The target protein originality was established by immunoblotting and electrophoresis; the correspondence of its primary structure to the declared sequence was demonstrated by mass-spectrophotometry.