2State Research Institute of Genetics and Selection of Industrial Microorganisms, 117545 Moscow, Russia
3Vernadsky Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, 119991 Moscow, Russia
4Sechenov Moscow State Medical University, 119991 Moscow, Russia
5Mental Health Research Center, 115522 Moscow, Russia
6All-Russia Research Institute of Agricultural Biotechnology, 127550 Moscow, Russia
7Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia
* To whom correspondence should be addressed.
Received July 4, 2017; Revision received August 3, 2017
Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture.
KEY WORDS: erythropoietin, heterologous expression, Escherichia coli