CRCU

Chemical Research in Chinese Universities ›› 2010, Vol. 26 ›› Issue (4): 591-595.

• Articles • Previous Articles     Next Articles

Purification and Characterization of Protein Tyrosine Phosphatase MEG1 and Preparation of Anti-PTPMEG1 Antibody

ZHANG Xiao-ping2, XING Shu1, WU Xiao-xia3, LIN Fan4, FU Xue-qi1* and LI Wan-nan1*   

  1. 1. Edmond H. Fischer Signal Transduction Laboratory, College of Life Sciences, Jilin University, Changchun 130012, P. R. China;
    2. Clinical Laboratory, China Japan Union Hospital, Jilin University, Changchun 130033, P. R. China;
    3. Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, Jilin University, Changchun 130012, P. R. China;
    4. School of Life Science, Northeast Normal University, Changchun 130024, P. R. China
  • Received:2009-09-30 Revised:2009-10-12 Online:2010-07-25 Published:2010-10-01
  • Contact: FU Xue-qi, E-mail: fxq@mail.jlu.edu.cn; LI Wan-nan, E-mail: liwannan@jlu.edu.cn
  • Supported by:

    Supported by the Plan of Development for Science & Technology of Jilin Province, China(No.20090920).

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Abstract: PTPMEG1 is an intracellular protein tyrosine phosphatase(PTP), which contains FERM and PDZ domains. This study focuses our attention on the expression, purification and characterization of catalytic domain of PTPMEG1(DMEG1) and preparation of its polyclonal antibody. A cDNA fragment encoding DMEG1 protein(amino acid residues 643―926) was amplified by PCR and then cloned into the pT7-7 vector. Both soluble and insoluble recombinant DMEG1 proteins were observed after induction by IPTG. Soluble DMEG1 was purified via two chromatographic steps, and the purified enzyme was characterized. With para-nitrophenylphosphate(pNPP) as a substrate, DMEG1 exhibited typical enzymatic characteristics of classic PTPs and classical Michaelis-Menten kinetics. Insoluble DMEG1, which was mainly distributed in the inclusion body of E. coli cells extracts, was purified by preparative electrophoresis gel for the preparation of the polyclonal antibody. A rabbit was immunized with DMEG1 purified by preparative electrophoresis to generate anti-DMEG1 antibody. Anti-serum was collected on 28th day after initial injection and purified via affinity chromatography. The purified polyconal antibody displayed a satisfactory titer and sensitivity.

Key words: Protein tyrosine phosphatase(PTP), PTPMEG1, DMEG1, Polyclonal antibody