Phosphatase of Regenerating Liver-3 （PRL-3） is a newly identified colorectal cancer metastasis-related protein, which is a 22 kDa non-classical protein tyrosine phosphatase with a C-terminal prenylation motif. In this study, the inhibition kinetics of protein tyrosine phosphatases（PTPs） by a fluorescent substrate, 6,8-difluoro-4-methylumbelliferyl phosphate（DiFMUP） was evaluated. PRL-3 exhibits classical Michaelis-Menten kinetics with a Vmax value of 11.3 μmol · L^-l · min^-1 Analysis of PRL-3 by a Michaelis-Menten plot and a double-reciprocal plot indicated that the inhibitor magnolol can cause Km to increase, but does not alter the Vmax value, which suggests the competitive inhibition of PRL-3. At the same time, it was found that DiFMUP is a more sensitive substrate for PRL-3 than para-nitrophenyl phosphate（pNPP） that is more frequently used at present. Furthermore, the method of screening for PTPs by the use of DiFMUP was developed, which studied the acceptance of DiFMUP by other PTPs.
Phosphatase of regenerating liver 3 （ PRL3 ）, which belongs to the superfamily of protein tyrosine phosphatases （PTPs）, represents a group of low molecular weight PTPs that participate in tumorigenesis and metastasis processes. Presented here are the results of cloning, prokaryotic expression, purification, and polyclonal antibody preparation of PRL3. To obtain a specific polyclonal antibody against PRL3, the authors have prepared GST-PRL3 to immunize rabbits and purify an anti-PRL3 polyclonal antibody by negative selection affinity columns. Western blot analysis shows that the anti-PRL3 polyclonal antibody has a specific binding ability with PRL3 protein. The anti-PRL3 polyclonal antibody provides a good tool to further study the function of PRL3.
SHEN Xing-gui LI Wan-nan WANG Jing JIANG Yi-qun LI Qing-shan FU Xue-qi
SPAP2,a transmembrane protein,is an Ig family receptor containing both ITIMs(immunoreceptor tyrosine-based inhibition motifs) and ITAMs(immunoreceptor tyrosine-based activation motifs).The extracellular portion of SPAP2 contains six immunoglobulin-like domains and its intracellular segment has two ITAMs and two ITIMs.Sequence alignment with the genomic database reveals that the SPAP2 gene contains 16 exons and is localized at chromosome 1q21.SPAP2 is consisted of 734 amino acids,and the intercellular portion of SPAP2 contains 137 amino acids. Tyrosine-phosphorylated SPAP2 is specifically associated with SH2 domain-containing tyrosine kinases and SH2 domain-containing tyrosine phosphatases,which lead to the initiation of signal transduction.SPAP2CT gene was amplified by PCR with SPAP2 full-length DNA as the template and cloned to the pBluescript Ⅱ KS vector.pGex-2T-SPAP2CT,the expression vector of dissoluble fusion protein,was constructed and transferred into E.coli of DE3-plysS.The fusion protein GST-SPAP2CT was expressed efficiently and purified by FFQ ion exchange chromatography and GSH affinity chromatography.The result indicates that we have constructed steady expression vector pGex-2T-SPAP2CT, which was expressed in E.coli.The molecular weight of the dissoluble fusion protein is 46 000,the productivity of GST-SPAP2CT protein is 10% and the purity is over 90% after the purification.
