Rat acid phosphatase: overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization.
Rat prostatic acid phosphatase (rPAP; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was expressed in the baculovirus expression vector system. Recombinant protein was secreted into the medium at a high yield by infected insect cells, which were cultured at high density in a 30-liter bioreactor allowing high oxygen content for rapidly growing cells. About 20% of the cell protein produced was rPAP. Partial sequence determination of the N terminus of the purified recombinant secreted protein revealed identity to the native secreted protein, showing that the signal peptide is recognized and properly cleaved in insect cells. The enzyme was purified by using L-(+)-tartrate affinity chromatography. The purified protein had a high specific activity of 2620 mumol.min-1.mg-1 with p-nitrophenyl phosphate at the substrate, and it also showed phosphotyrosine phosphatase activity. The molecular mass of the recombinant rPAP was 155 kDa. Two subunits of 46 kDa and 48 kDa could be detected in SDS/PAGE, but only one subunit of 41 kDa was present after digestion with N-glycosidase. The active enzyme is a trimer of subunits differing only in glycosylation. When recombinant rPAP was crystallized with polyethylene glycol 6000 as the precipitant, the crystals were trigonal (space group P3(1)21) with cell dimensions a = 89.4 A and c = 152.0 A. The observed diffraction pattern extends to a resolution of at least 3 A.
Vihko
P
P
Department of Clinical Chemistry, University of Oulu, Finland.
Kurkela
R
R
Porvari
K
K
Herrala
A
A
Lindfors
A
A
Lindqvist
Y
Y
Schneider
G
G
D000135:Acid Phosphatase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D003460:Crystallization; D008297:Male; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D011467:Prostate / Q000201:enzymology*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014961:X-Ray Diffraction
D011994:Recombinant Proteins; D000135:Acid Phosphatase