Abstract
A gene (No. AF0497 GenBank, USA) was cloned from the archaeon Archaeoglobus fulgidus strain found in the water of hot springs. This gene contains an open reading frame of 2346 base pairs which encodes a thermostable DNA-polymerase (762 amino acid residues). A recombinant protein Afu-pol with molecular weight of 94 kD was isolated in an Escherichia coli strain used as a producer and characterized. By site-directed mutagenesis in the afu-pol gene the amino acid residue Glu170 was replaced with Ala; this resulted in a complete loss of the 3"-5"-exonuclease activity of the enzyme. Thus, the Glu170 residue was suggested to be directly involved in formation of the 3"-5"-exonuclease site. Physicochemical features of the exodeficient enzyme form were studied, and the possible use of Afu(exo–)-pol in the polymerase chain reaction is shown.
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REFERENCES
Braithwaite, D. K., and Ito, J. (1993) Nucleic Acids Res., 21, 787-802.
Li, Y., Korolev, S., and Waksman, G. (1998) EMBO J., 17, 7514-7525.
Hopfner, K. P., Eichinger, A., Engh, R. A., Laue, F., Ankenbauer, W., Huber, R., and Angerer, B. (1999) Proc. Natl. Acad. Sci. USA, 96, 3600-3605.
Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A. (1988) Proc. Natl. Acad. Sci. USA, 85, 9436-9440.
Venter, J. C., et al. (1997) Nature, 390, 364-370.
Ishino, Y., Komori, K., Cann, I. K., and Koga, Y. (1998) J. Bacteriol., 180, 2232-2236.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, Cold Spring Harbor, N. Y.
Scharf, S. G. (1990) Cloning with PCR, Academic Press, San Diego, California.
Protocols and Application Guide (1991), Promega, pp. 84-87.
Lawyer, F. C., Stoffel, S., Saiki, R. K., Myambo, K., Drummond, R., and Gelfand, D. H. (1989) J. Biol. Chem., 264, 6427-6437.
Laemmli, U. K. (1979) Nature, 227, 680-685.
Wang, J., Sattar, A. K., Wang, C. C., Karam, J. D., Konigsberg, W. H., and Steitz, T. A. (1997) Cell, 89, 1087-1099.
Joyce, C. M., and Steitz, T. A. (1994) Ann. Rev. Biochem., 63, 777-822.
Dang, C., and Jayasena, S. D. (1996) J. Mol. Biol., 264, 268-278.
Blanco, L., Salas, M., et al. (1991) Gene, 100, 27-38.
Southworth, M. W., Kong, H., Kucera, R. B., Ware, J., Jannasch, H. W., and Perler, F. B. (1996) Proc. Natl. Acad. Sci. USA, 93, 5281-5285.
Perler, F. B., Kumar, S., and Kong, H. (1996) Adv. Protein Chem., 48, 377-435.
Kong, H., Kucera, R. B., and Jack, W. E. (1993) J. Biol. Chem., 268, 1965-1975.
Harald, H. (1998) J. Biotechnol., 64, 39-52.
Korolev, S., Nayal, M., Barnes, W. M., Di Cera, E., and Waksman, G. (1995) Proc. Natl. Acad. Sci. USA, 92, 9264-9268.
Tagaki, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B., Oka, M., and Imanaka, T. (1997) Appl. Environ. Microbiol., 63, 4504-4510.
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Chalov, S.E., Voronina, O.L., Sergienko, O.N. et al. Thermostable DNA-Polymerase from the Thermophilic Archaeon Microorganism Archaeoglobus fulgidus VC16 and Its Features. Biochemistry (Moscow) 68, 301–308 (2003). https://doi.org/10.1023/A:1023054200483
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DOI: https://doi.org/10.1023/A:1023054200483