Abstract
Twenty-nine aminotransferase genes from Pyrococcus horikoshii, Aeropyrum pernix, and Sulfolobus tokodaii were cloned and expressed in Escherichia coli. The expression of several of the genes at 15, 25, or 37 °C resulted in the formation of insoluble protein aggregates. Therefore, we developed a simple method to express these genes into soluble proteins, by cultivating E. coli clones at a higher temperature. Thus, four genes could be expressed efficiently into soluble and active enzymes by cultivating the respective E. coli clones at 46 °C. Subsequently, the method was applied to the expression into soluble proteins of other aminotransferase genes that were derived from nine species of thermophilic microorganisms.
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References
Andreotti G, Cubellis MV, Nitti G, Sannia G, Mai X, Adams MWW, Marino G (1995) An extremely thermostable aromatic aminotransferase from the hyperthermophilic archaeon Pyrococcus furiosus. Biochim Biophys Acta 1247:90–96
Andronopoulou E, Vorgias CE (2004) Isolation, cloning, and overexpression of a chitinase gene fragment from the hyperthermophilic archaeon Thermococcus chitonophagus: semi-denaturing purification of the recombinant peptide and investigation of its relation with other chitinases. Protein Expr Purif 35:264–271
Bowden GA, Georgiou G (1990) Folding and aggregation of beta-lactamase in the periplasmic space of Escherichia coli. J Biol Chem 265:16760–16766
Dabrowski S, Kiaer AB (2003) Cloning, expression, and purification of the His6-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR. Protein Expr Purif 31:72–78
Grzybowska B, Szweda P, Synowiecki J (2004) Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme. Mol Biotechnol 26:101–110
Hannig G, Makrides SC (1998) Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnol 16:54–60
Humphreys DP, Sehdev M, Chapman AP, Ganesh R, Smith BJ, King LM, Glover DJ, Reeks DG, Stephens PE (2000) High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5′ end of the coding sequence. Protein Expr Purif 20:252–264
Ideno A, Furutani M, Iwabuchi T, Iida T, Iba Y, Kurosawa Y, Sakuraba H, Ohshima T, Kawarabayashi Y, Maruyama T (2004) Expression of foreign proteins in Escherichia coli by fusing with an archaeal FK506 binding protein. Appl Microbiol Biotechnol 64:99–105
Ishida M, Oshima T, Yutani K (2002) Overexpression in Escherichia coli of the AT-rich trpA and trpB genes from the hyperthermophilic archaeon Pyrococcus furiosus. FEMS Microbiol Lett 216:179–183
Jespers L, Sonveaux E, Fastrez J, Phanapoulos A, Davison J (1991) Overexpression of the phage lambda lysozyme cloned in Escherichia coli: use of a degenerative mixture of synthetic ribosome binding sites and increase of the protein stability in vivo. Protein Eng 4:485–492
Kim BC, Lee YH, Lee HS, Lee DW, Choe EA, Pyun YR (2002) Cloning, expression and characterization of l-arabinose isomerase from Thermotoga neapolitana: bioconversion of d-galactose to d-tagatose using the enzyme. FEMS Microbiol Lett 212:121–126
Kochhar S, Mouratou B, Christen P (2000) Amino acid analysis by high-performance liquid chromatography after derivatization with 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide (Marfey’s reagent). Methods Mol Biol 159:49–54
Kopetzki E, Schumacher G, Buckel P (1989) Control of formation of active soluble or inactive insoluble baker’s yeast alpha-glucosidase PI in Escherichia coli by induction and growth conditions. Mol Gen Genet 216:149–155
Lu C, Erickson HP (1997) Expression in Escherichia coli of the thermostable DNA polymerase from Pyrococcus furiosus. Protein Expr Purif 11:179–184
Marfey P (1984) Determination of d-amino acids. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res Commun 49:591–596
Moreno R, Haro A, Castellanos A, Berenguer J (2005) High-level overproduction of His-tagged Tth DNA polymerase in Thermus thermophilus. Appl Environ Microbiol 71:591–593
Nishihara K, Kanemori M, Kitagawa M, Yanagi H, Yura T (1998) Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli. Appl Environ Microbiol 64:1694–1699
Novy R, Drott D, Yaeger K, Mierendorf R (2001) Overcoming the codon bias of E. coli for enhanced protein expression. Innovations 12:1–3
Ogasahara K, Nakamura M, Nakura S, Tsunasawa S, Kato I, Yoshimoto T, Yutani K (1998) The unusually slow unfolding rate causes the high stability of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus: equilibrium and kinetic studies of guanidine hydrochloride-induced unfolding and refolding. Biochemistry 37:17537–17544
Sambrock J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Scalley ML, Baker D (1997) Protein folding kinetics exhibit an Arrhenius temperature dependence when corrected for the temperature dependence of protein stability. Proc Natl Acad Sci USA 94:10636–10640
Schofield LR, Patchett ML, Parker EJ (2004) Expression, purification, and characterization of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase from Pyrococcus furiosus. Protein Expr Purif 34:17–27
Word DE, Kengen SWM, van Der Oost J, de Vos WM (2000) Purification and characterization of the alanine aminotransferase from the hyperthermophilic archaeon Pyrococcus furiosus and its role in alanine production. J Bacteriol 182:2559–2566
Yu L, Deng K-P, Yu C-A (1995) Cloning, gene sequencing, and expression of the small molecular mass ubiquinone-binding protein of mitochondrial ubiquinol-cytochrome c reductase. J Biol Chem 270:25634–25638
Acknowledgements
This study was supported in part by the 21C-COE Program “Practical Nano-Chemistry” from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan and by a Waseda University Grant for Special Research Projects (2005B-142). We are grateful to Dr. Katsumi Isono for the critical review and helpful discussions.
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Koma, D., Sawai, T., Harayama, S. et al. Overexpression of the genes from thermophiles in Escherichia coli by high-temperature cultivation. Appl Microbiol Biotechnol 73, 172–180 (2006). https://doi.org/10.1007/s00253-006-0448-9
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DOI: https://doi.org/10.1007/s00253-006-0448-9