Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean.
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.
Vogel
Andreas
A
Department of Biochemistry, University of Münster, Wilhelm-Klemm-Strasse 2, 48149 Münster, Germany.
Börchers
Torsten
T
Marcus
Katrin
K
Meyer
Helmut E
HE
Krebs
Bernt
B
Spener
Friedrich
F
D000135:Acid Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D027805:Phaseolus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D018076:DNA, Complementary; D018744:DNA, Plant; D006020:Glycopeptides; D006023:Glycoproteins; D011994:Recombinant Proteins; C091137:purple acid phosphatase; D000135:Acid Phosphatase