Cloning and expression of the bovine intestinal alkaline phosphatase gene: biochemical characterization of the recombinant enzyme.
A complete genomic clone and a full-length cDNA coding for bovine intestinal alkaline phosphatase have been isolated and sequenced. The gene (5.4 kb) contains 11 exons separated by ten small introns at positions identical to those other members of the eukaryotic tissue-specific alkaline phosphatase family. In addition, 1.5 kb of upstream sequences contain putative regulatory elements showing sequence similarity to human and mouse intestinal alkaline phosphatase promoter sequences. To achieve recombinant bovine intestinal alkaline phosphatase expression, the coding region of the gene was subcloned into the pcDNA I eukaryotic expression vector and transfected into Chinese hamster ovary cells. Recombinant bovine intestinal alkaline phosphatase displays enzymatic properties comparable with those of purified native bovine intestinal alkaline phosphatase, a slightly increased thermal stability and, upon desialylation, it shows a homogeneous behaviour in agarose gel electrophoresis and isoelectric focusing. The availability of the recombinant bovine intestinal alkaline phosphatase and the elucidation of its primary sequence will help to accelerate our efforts to obtain the first crystallographic model of a eukaryotic alkaline phosphatase molecule.
Weissig
H
H
La Jolla Cancer Research Foundation, Cancer Research Center, CA 92037.
Schildge
A
A
Hoylaerts
M F
MF
Iqbal
M
M
Millán
J L
JL
D000469:Alkaline Phosphatase / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002417:Cattle; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA; D007422:Intestines / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D004247:DNA; D000469:Alkaline Phosphatase