Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in Escherichia coli heterologous...
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.
Biochemistry (Moscow)
Vol.82, Issue11, P. 1285-1294
Опубликовано: 2017
Тип ресурса: Статья
DOI:10.1134/S0006297917110062
Аннотация:
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
Ключевые слова:
erythropoietin; Escherichia coli; heterologous expression
enteropeptidase; EPO protein, human; erythropoietin; His-His-His-His-His-His; histidine; hybrid protein; maltose binding protein; oligopeptide; pancreatic ribonuclease; peptide fragment; biosynthesis; chemistry; chromatography; Escherichia coli; gene expression; genetics; human; isolation and purification; metabolism; protein conformation; protein domain; Chromatography; Enteropeptidase; Erythropoietin; Escherichia coli; Gene Expression; Histidine; Humans; Maltose-Binding Proteins; Oligopeptides; Peptide Fragments; Protein Conformation; Protein Domains; Recombinant Fusion Proteins; Ribonuclease, Pancreatic
Язык текста: Английский
ISSN: 1608-3040
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.
Грунина Т. М.
Демиденко А. В.
Лясчук А. М.
Попонова М. С.
Галушкина З. М.
Соболева Л. А.
Черепушкин С. А.
Поляков Н. Б.
Грумов Д. А.
Соловьев А. И.
Жуховицкy В. Г.
Бокша И. С.
Субботина М. Е.
Громов А. В.
Лунин В. Г.
Карягина А. С.
Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in Escherichia coli heterologous expression system
Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in Escherichia coli heterologous...
Текст визуальный непосредственный
Biochemistry (Moscow)
Pleiades Publishing, Ltd.
Vol.82, Issue11 P. 1285-1294
2017
Статья
erythropoietin Escherichia coli heterologous expression
enteropeptidase EPO protein, human erythropoietin His-His-His-His-His-His histidine hybrid protein maltose binding protein oligopeptide pancreatic ribonuclease peptide fragment biosynthesis chemistry chromatography Escherichia coli gene expression genetics human isolation and purification metabolism protein conformation protein domain Chromatography Enteropeptidase Erythropoietin Escherichia coli Gene Expression Histidine Humans Maltose-Binding Proteins Oligopeptides Peptide Fragments Protein Conformation Protein Domains Recombinant Fusion Proteins Ribonuclease, Pancreatic
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