title: Chaperones are necessary for the expression of catalytically active potato apyrases in prokaryotic cells.
abstract: NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5'-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.
authors.affiliation: Biochemistry Department, Faculty of Biology and Environment Protection, Nicolaus Copernicus University in Torun, Torun, Poland, dorota.porowinska@cm.umk.pl.
authors.forename: Dorota
authors.initials: D
authors.lastname: Porowińska
authors.forename: Joanna
authors.initials: J
authors.lastname: Czarnecka
authors.forename: Michał
authors.initials: M
authors.lastname: Komoszyński
mesh_terms: D001081:Apyrase / Q000096:biosynthesis* / Q000235:genetics*; D001709:Biotechnology; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017343:Genes, Plant; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics*
chemical_list: D018835:Chaperonin 10; D018834:Chaperonin 60; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D010940:Plant Proteins; D011994:Recombinant Proteins; C063842:dnaK protein, E coli; D001081:Apyrase