Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in Escherichia coli heterologous expression system

• Published in: Biochemistry (Moscow) 2017-11, Vol.82 (11), p.1285-1294
• Main Authors: Grunina, T. M., Demidenko, A. V., Lyaschuk, A. M., Poponova, M. S., Galushkina, Z. M., Soboleva, L. A., Cherepushkin, S. A., Polyakov, N. B., Grumov, D. A., Solovyev, A. I., Zhukhovitsky, V. G., Boksha, I. S., Subbotina, M. E., Gromov, A. V., Lunin, V. G., Karyagina, A. S.
• Format: Article
• Language: English
• Subjects: Biochemistry; Biological activity; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Cell culture; Chromatography; Cleavage; Column chromatography; Disulfide bonds; E coli; Enteropeptidase - metabolism; Erythropoietin; Erythropoietin - biosynthesis; Erythropoietin - genetics; Erythropoietin - isolation & purification; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Genetic aspects; Genetic recombination; Health aspects; Histidine; Humans; Kinases; Life Sciences; Maltose; Maltose-binding protein; Maltose-Binding Proteins - chemistry; Maltose-Binding Proteins - genetics; Mass spectrometry; Mass spectroscopy; Microbiology; Oligopeptides; Pancreas; Pancreatic ribonuclease; Peptide Fragments; Protein Conformation; Protein Domains; Protein purification; Protein synthesis; Proteolysis; Purification; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Red blood cells; Ribonuclease A; Ribonuclease, Pancreatic - chemistry; Terminal protein; Yeast
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1134/S0006297917110062

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.