Protein tyrosine phosphorylation is one of the important mechanisms of signal transduction in cells. The level is regulated by the balanced action of protein tyrosine kinases and protein tyrosine phosphatases.
To obtain the molecular tools for further study of the biological function of protein tyrosine phosphatases in mouse, I have cloned a protein tyrosine phosphatase gene from mouse brain employing radioisotope-labeled probe of human placental protein tyrosine phosphatase 1B. A plaque showing strong positive signal was isolated from a mouse brain cDNA library, subcloned into pUC19 and determined its nucleotide sequence. The isolated clone (MBPTP1B) was found to contain an open reading frame of 1,296 nucleotides as well as 5' (709 nucleotides) and 3' (341 nucleotides) non-coding regions. This cDNA encodes a PTP of 432 amino acids having a mass of 49,563 daltons and exhibiting 83% and 93.5% sequence identity to that of human PTP1B and rat PTP1, respectively. Therefore, it is suggested that MBPTP1B is one of non-receptor type PTPs in mouse and a mouse counterpart of HPTP1B and RPTP1.
To express the coding sequence of MBPTP1B cDNA in yeast, pYEPTP, yeast-E. coli shuttle vector containing GAP promoter and GAL7 transcription terminator, was constructed. The expression vector was transformed into Saccharomyces diastaticus YIY345. The MBPTP1B was expressed from the yeast harboring pYEPTP in a small quantity. So, western blot analysis using anti CPTP1 antibody was carried out for the identification of MBPTP1B expression in the yeast. The result of PTP activity assay using p-nitrophenyl phosphate as artificial substrate also showed that MBPTP1B is a PTP having PTP activity. MBPTP1B had the optimum activity at 37 ℃ and pH 7.5 as the case of many enzymes in mouse. MBPTP1B was inhibited by sodium orthovanadate, a potent PTP inhibitor, but not by okadaic acid known as a potent serine/threonine phosphatase inhibitor.
To prepare antibody for the C-terminal region (135 amino acid residues) of MBPTP1B, pMAL-CT expression vector as MBP fusion protein was constructed. The vector was transformed into E. coli JM109 and the expression of MBP-CT was identified as expected mass (57 kDa) by SDS-PAGE. The anti MBP-CT antibody obtained from rabbit was shown to react with MBP, MBP-CT, MBPTP1B and HPTP1B by western blot analysis. Thus the antibody will be able to be used in further PTP-related research.
