Abstract
Polyhistidine-tagged dihydrofolate synthase (DHFS) has been produced in the yeast, Saccharomyces cerevisiae, using a Cu2+-inducible expression system. The tagged DHFS is functional in vivo and was purified using immobilised metal affinity chromatography. A linker of a minimal size allows efficient cleavage of the poly-His tag using thrombin. At least 10 mg of pure DHFS can be recovered per litre of culture.
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References
Alleva JL, Doetsch PW (1998) Characterization of Schizosaccharomyces pombe Rad2 protein, a FEN-1 homolog. Nucl. Acids Res. 26: 3645-3650.
Arunagiri CK, Macreadie IG, Hewish DR, Azad AA (1997) A membrane-active C-terminal domain of HIV-1 accessory protein Vpr penetrates human cells and causes mitochondrial dysfunction and apoptosis. Apoptosis 2: 69-76.
Bayly AM, Berglez JM, Patel O, Castelli LA, Hankins EG, Coloe P, Hopkins Sibley C, Macreadie IG (2001) Folic acid utilisation related to sulfa drug resistance in Saccharomyces cerevisiae. FEMS Microbiol. Lett. 199: 184-194.
Castelli LA, Petris AJ, Carroll SM, Macreadie IG (1995) Secretion and purification of glutathione S-transferase fusion proteins from yeast. Biotechnol. Tech. 8: 829-826.
Chang JY (1985) Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate. Eur. J. Biochem. 151: 217-224.
Chen HM, Luo SL, Chen KT, Lii CK (1999) Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography. J. Chromatogr. A852: 151-159.
D'Alatri L, Di Massimo AM, Anastasi AM, Pacilli A, Novelli S, Saccinto MP, De Santis R, Mele A, Parente D (1998) Production and characterisation of a recombinant single-chain anti ErbB2-clavin immunotoxin. Anticancer Res. 18: 3369-3373.
Enomoto S, Chen G, Berman J (1998) Vectors for expressing T7 epitope-and His6 affinity-tagged fusion proteins in S. cerevisiae. Biotechniques 24: 782-788.
Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SSDNA/ PEG procedure. Yeast 11: 355-360.
Guan KL, Dixon JE (1991) Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192: 262-267.
Laemmli UK, Favre M (1973) Maturation of the head of bacteriophage T4. I. DNA packaging events. J. Mol. Biol. 80: 575-599.
Macreadie IG, Vaughan PR (1998) Development of yeast expression systems. In: Cremonesi P, Yoshida H, Mori K, Haruhito T, eds., Recent Research Developments in Biotechnology and Bioengineering, pp. 465-479.
Macreadie IG, Castelli LA, Hewish DR, Kirkpatrick A, Ward AC, Azad AA (1995) A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects. Proc. Natl. Acad. Sci. USA 92: 2770-2774.
Macreadie IG, Failla P, Horaitis O, Azad AA (1992) Production of HIV-1 Vpu with pYEULCBX, a convenient vector for the production of non-fused proteins in yeast. Biotechnol. Lett. 14: 639-642.
Macreadie IG, Horaitis O, Verkuylen AJ, Savin KW (1991) Improved shuttle vectors for cloning and high-level Cu2+-mediated expression of foreign genes in yeast. Gene 104: 107-111.
Negro A, Onisto M, Masiero L, Garbisa S (1995) Synthesis and refolding of human TIMP-2 from E. coli, with specific activity for MMP-2. FEBS Lett. 360: 52-56.
Svensson J, Palva ET, Welin B (2000) Purification of recombinant Arabidopsis thaliana dehydrins by metal ion affinity chromatography. Protein Expr. Purif. 20: 169-178.
Ward AC, Azad AA, Macreadie IG (1994a) Expression and characterisation of the influenza A virus non-structural protein NS1 in yeast. Arch. Virol. 138: 299-314.
Ward AC, Castelli LA, Macreadie IG, Azad AA (1994b) Vectors for Cu2+-inducible production of glutathione S-transferase fusion proteins for single-step purification from yeast. Yeast 10: 441-449.
Zouhar J, Nanak E, Brzobohaty B (1999) Expression, singlestep purification, and matrix-assisted refolding of a maize cytokinin glucoside-specific beta-glucosidase. Protein Expr. Purif. 17: 153-162.
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Patel, O., Castelli, L., Fernley, R. et al. Production of a functional dihydrofolate synthase with a cleavable poly-His tag in Saccharomyces cerevisiae . Biotechnology Letters 24, 657–662 (2002). https://doi.org/10.1023/A:1015051526401
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DOI: https://doi.org/10.1023/A:1015051526401