Recombinant alpha-fetoprotein receptor-binding domain co-expression with polyglutamate tags facilitates in vivo folding in E. coli
Mollaev M., Gorokhovets N. V., Nikolskaya E., Faustova M., Zabolotsky A., Sokol M., Tereshenko O., Zhunina O., Shvets V., Severin E. S., Yabbarov N.
Protein Expression and Purification
Vol.143, P. 77-82
Опубликовано: 2018
Тип ресурса: Статья
DOI:10.1016/j.pep.2017.11.001
Аннотация:
A wide range of methods are known to increase the prokaryotic intracellular recombinant proteins solubility, for instance, growth at low temperature, supplementation of culture media with “chemical chaperones” (proline, glycine-betaine, and trehalose), co-expression with chaperones or highly soluble fusion partners. As an alternative, we have introduced the polyglutamate tag, which, as it has been shown, increased the protein solubility and facilitated folding. In this study we evaluated the minimal quantity of high density negatively charged EEEEVE amino acid repeats (pGlu) necessary to switch the recombinant receptor-binding domain of human alpha-fetoprotein (rbdAFP) expression almost entirely from the inclusion bodies to the soluble cytoplasmic fraction in E. coli. For this purpose, genetic constructs based on pET vectors coding rbdAFP and containing from 1 to 4 additional EEEEVE repeats at the C-terminus have been prepared. It was found that 3 pGlu repeats is the minimal number, th
Ключевые слова:
Alpha-fetoprotein receptor-binding domain; E. coli in vivo protein folding; EEEEVE; pGlu tag; rbdAFP; Soluble protein expression
alpha-fetoprotein receptor, human; fusion protein; polyglutamic acid; receptor; cell culture; chemistry; Escherichia coli; genetics; human; isolation and purification; metabolism; mononuclear cell; protein domain; protein folding; tumor cell line; Cell Line, Tumor; Cells, Cultured; Escherichia coli; Humans; Leukocytes, Mononuclear; Polyglutamic Acid; Protein Domains; Protein Folding; Receptors, Peptide; Recombinant Fusion Proteins
Язык текста: Английский
ISSN: 1096-0279
Mollaev M.
Gorokhovets N. V. Neonila Vasilyevna 1956-
Nikolskaya E.
Faustova M.
Zabolotsky A.
Sokol M.
Tereshenko O.
Zhunina O.
Shvets V.
Severin E. S. Evgenij Sergeevich 1934-
Yabbarov N.
Моллаев М.
Гороховец Н. В. Неонила Васильевна 1956-
Николскайа Е.
Фаустова М.
Заболоцкy А.
Сокол М.
Терешенко О.
Жунина О.
Швец В.
Северин Е. С. Евгений Сергеевич 1934-
Яббаров Н.
Recombinant alpha-fetoprotein receptor-binding domain co-expression with polyglutamate tags facilitates in vivo folding in E. coli
Текст визуальный непосредственный
Protein Expression and Purification
Academic Press
Vol.143 P. 77-82
2018
Статья
Alpha-fetoprotein receptor-binding domain E. coli in vivo protein folding EEEEVE pGlu tag rbdAFP Soluble protein expression
alpha-fetoprotein receptor, human fusion protein polyglutamic acid receptor cell culture chemistry Escherichia coli genetics human isolation and purification metabolism mononuclear cell protein domain protein folding tumor cell line Cell Line, Tumor Cells, Cultured Escherichia coli Humans Leukocytes, Mononuclear Polyglutamic Acid Protein Domains Protein Folding Receptors, Peptide Recombinant Fusion Proteins
A wide range of methods are known to increase the prokaryotic intracellular recombinant proteins solubility, for instance, growth at low temperature, supplementation of culture media with “chemical chaperones” (proline, glycine-betaine, and trehalose), co-expression with chaperones or highly soluble fusion partners. As an alternative, we have introduced the polyglutamate tag, which, as it has been shown, increased the protein solubility and facilitated folding. In this study we evaluated the minimal quantity of high density negatively charged EEEEVE amino acid repeats (pGlu) necessary to switch the recombinant receptor-binding domain of human alpha-fetoprotein (rbdAFP) expression almost entirely from the inclusion bodies to the soluble cytoplasmic fraction in E. coli. For this purpose, genetic constructs based on pET vectors coding rbdAFP and containing from 1 to 4 additional EEEEVE repeats at the C-terminus have been prepared. It was found that 3 pGlu repeats is the minimal number, th