Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Vogel, A; Börchers, T; Marcus, K; Meyer, HE; Krebs, B; Spener, F.
Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean.
Arch Biochem Biophys. 2002; 401(2):164-172
Doi: 10.1016/S0003-9861(02)00046-2
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- Führende Autor*innen der Med Uni Graz
- Spener Friedrich
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- Abstract:
- Purple acid phosphatases (PAPs) are dinuclear metallohydrolases of widespread occurrence. In a first step to understand structure-function relationship of PAP from red kidney bean (kbPAP), we cloned its cDNA and functionally expressed the enzyme in insect cells. kbPAP cDNA encodes a protein of 459 amino acids with 99% identity to the published primary structure (T. Klabunde et al., Eur. J. Biochem. 226 (1994) 369-375). N-terminally the cDNA encodes 27 amino acids with characteristics for a signal directing the nascent protein to the endoplasmic reticulum. A baculovirus vector was constructed containing cDNAs of kbPAP and green fluorescent protein, the latter to serve as transfection and infection marker. Heterologous expression in High Five insect cells afforded a dimeric, disulfide-linked phosphatase of 110 kDa, identical to the mass of native kbPAP. Purification in three steps yielded 1.5 mg recombinant protein per liter of culture medium with a specific activity of 266 units/mg, slightly exceeding that of native kbPAP. The recombinant protein was functionally indistinguishable from native kbPAP, despite differences in glycosylation and sensitivity to redox reagents.
- Find related publications in this database (using NLM MeSH Indexing)
- Acid Phosphatase - chemistry Acid Phosphatase - genetics Acid Phosphatase - metabolism
- Amino Acid Sequence -
- Animals -
- Baculoviridae - genetics
- Base Sequence -
- Cell Line -
- Cloning, Molecular -
- DNA, Complementary - genetics
- DNA, Plant - genetics
- Escherichia coli - genetics
- Gene Expression -
- Genes, Plant -
- Glycopeptides - chemistry
- Glycoproteins - chemistry Glycoproteins - genetics Glycoproteins - metabolism
- Molecular Sequence Data -
- Phaseolus - enzymology Phaseolus - genetics
- Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism
- Spodoptera -
- Find related publications in this database (Keywords)
- purple acid phosphatase
- plant
- tartrate-resistant acid phosphatase
- metalloenzyme
- heterologous expression
- baculovirus