Cloning and expression of a protein-tyrosine-phosphatase.
A rat brain cDNA library was screened by using a mixture of oligonucleotides whose sequences were deduced from the amino acid sequence of a human placental protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) reported by Charbonneau et al. [Charbonneau, H., Tonks, N. K., Walsh, K. A. & Fischer, E. H. (1988) Proc. Natl. Acad. Sci. USA 85, 7182-7186]. The isolated clones encode a PTPase of 432 amino acids having a mass of 49,679 daltons and showing 97% sequence identity to the corresponding 321 residues of the placental enzyme. The coding sequence of the PTPase was placed behind a bacteriophage T7 promoter and the protein was expressed in Escherichia coli. The recombinant protein had a molecular weight of 50,000 by SDS/PAGE analysis and showed an absolute specificity for phosphotyrosine-containing substrates. Northern analysis documented that there were two sizes of RNA, 4.3 and 2.0 kilobases, which encode the PTPase. Both transcripts were present in a number of tissues, and the smaller RNA appears to arise by the use of an alternative polyadenylylation signal. The PTPase was also localized by in situ hybridization in the rat central nervous system. A diffuse pattern of hybridization signal is seen in a number of brain areas, with the hippocampus showing the highest levels of mRNA. Sequences located at the C terminus of the rat brain PTPase contain possible sites for phosphorylation as well as signals which could serve for membrane attachment.
Guan
K L
KL
Department of Biochemistry, Purdue University, West Lafayette, IN 47907.
Haun
R S
RS
Watson
S J
SJ
Geahlen
R L
RL
Dixon
J E
JE
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D007031:Hypothalamus / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017027:Protein Tyrosine Phosphatases; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004247:DNA; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases