Cloning and expression of a yeast protein tyrosine phosphatase.
To study the regulation of tyrosine phosphorylation/dephosphorylation in Saccharomyces cerevisiae, a protein tyrosine phosphatase (PTPase) was cloned by the polymerase chain reaction (PCR). Conserved amino acid sequences within the mammalian PTPases were used to design primers which generated a yeast PCR fragment. The sequence of the PCR fragment encoded a protein with homology to the mammalian PTPases. The PCR fragment was used to identify the yeast PTP1 gene which has an open reading frame encoding a 335-amino acid residue protein. This yeast PTPase shows 26% sequence identity to the rat PTPase, although highly conserved residues within the mammalian enzymes are invariant in the yeast protein. The yeast PTP1 is physicallt linked to the 5'-end of a heat shock gene SSB1. This yeast PTP1 gene was expressed in Escherichia coli and obtained in a highly purified form by a single affinity chromatography step. The recombinant yeast PTPase hydrolyzed phosphotyrosine containing substrates approximately 1000 times faster than a phosphoserine containing substrate. Gene disruption of yeast PTP1 has no visible effect on vegetative growth.
Guan
K L
KL
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Deschenes
R J
RJ
Qiu
H
H
Dixon
J E
JE
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017027:Protein Tyrosine Phosphatases; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases