Recombinant Fusion E7 Oncoprotein of Human Papillomavirus Type 16 Conjugated with Heat Shock Protein 70: Isolation and Identification

Автор: M.A. Zhuchenko, N.A. Shamonov, M.V. Serebriakova, and S.A. Cherepushkin

Страница: 29-37

Recombinant Fusion E7 Oncoprotein of Human Papillomavirus Type 16 Conjugated with Heat Shock Protein 70: Isolation and Identification

Biotekhnologiya, 2015, N 1, P. 29-37

UDC 573.6.086.83:577.21:[615.373.3+615.277]

Section:  “Biologicals Technology”

M.A. Zhuchenko ^{1, *},  N.A. Shamonov ¹,  M.V. Serebriakova ^{1, 2},  and  S.A. Cherepushkin ¹

¹  The State Research Institute for Genetics and Selection of Industrial Microorganisms,  117545, Moscow Russia

²  The Belozerskii Researh Institute for Physicochemical Biology,  Moscow Russia

e-mail:  axiflipper@gmail.com

A gene for a recombinant E7 oncoprotein of human papillomavirus type 16 conjugated with a heat shock protein 70 (E7(16)-HSP70) has been expressed in Saccharomyces cerevisiae. A number of chromatographic separations was performed in order to primarily identify the protein and establish its authenticity. Metal-chelate and affinity chromatography were aimed at selective binding of the active groups of sorbents with polyhistidine fragment at the C-terminus of the polypeptide sequence of the target protein and ATP-binding regions of E7 and HSP70, respectively. The purity of the target protein after two purification steps did not exceed 70% showing the presence of a number of low-molecular impurities and oligomerization products of the target molecule. Chromatographic purification under denaturing conditions using strong reducing agents made it possible to isolate the target protein with the content of the monomer exceeding 97%. Electrophoresis and immunoblotting methods permitted to establish the authenticity of the target protein, and mass spectrometry showed its set primary structure.

Key words:  heat shock protein,  human papilloma virus,  vaccine.

29.05.2015, 1669 просмотров.