A Technique for Purification of Hybrid Recombinant E7 Oncoproteins of 16 and 18 Types of Human Papilloma Virus Conjugated with Heat Shock Protein70

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

Страница: 54-63

A Technique for Purification of Hybrid Recombinant E7 Oncoproteins of 16 and 18 Types of Human Papilloma Virus Conjugated with Heat Shock Protein70

Biotekhnologiya, 2015, N 3, P. 54-63

UDC 577.112.083

Section:  “Biologicals Technology”

M.A. Zhuchenko ^*,  N.A. Shamonov,  S.A. Cherepushkin,  and  N.A. Gavrilova

The State Research Institute for Genetics and Selection of Industrial Microorganisms (GosNIIgenetika),  117545, Moscow Russia

e-mail:  axiflipper@gmail.com

A nickel-chelate affinity chromatography has been used to isolate fused proteins of Hsp70-E7 from yeast lysate at the initial stage of the work. The low purity of the monomeric Hsp70-E7 in the final fraction was associated with the occurrence of dimeric and tetrameric forms of the target protein and low-molecular impurities. A modification of the purification procces by the addition of reduced L-glutathione to inhibit the formation of unwanted disulfide bonds permitted to increase the purity of the target protein by reducing the content of the oligomerized products. Further changes in the purification protocol, namely the reduction of hydrophobic interactions by the introduction of glycerol, significantly reduced the amount of low-molecular impurities. For a finer purification of E7-HSP70, a two-stage process of anion-exchange chromatography that made it possible to effectively eliminate low-molecular impurities was used. The assessment of the final material by electrophoresis under non-reducing conditions showed that the purity of the monomeric forms of either of the proteins was higher than 95%.

Key words:  chromatographic purification,  dimerization,  E7 oncoprotein,  heat shock protein.

07.09.2015, 1367 просмотров.