›› 2009, Vol. 29 ›› Issue (10): 1152-.

• Original article (Basic research) • Previous Articles     Next Articles

Construction, purification and substrate specificity identification of recombinant human platelet-activating factor acetylhydrolase isoformⅠ

CHEN Xiao-ying1, XU Jing2, YANG Jun-wei3, ZHANG Yi-xuan1   

  1. 1. School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China;2. Shanghai Institute of Health Sciences, Shanghai 201318, China;3. Institute of Plant Physiology and Ecology, Shanghai Institute for Biological Sciences, Shanghai 200032, China
  • Online:2009-10-25 Published:2009-10-26
  • Supported by:

    National Natural Science and Technology Resource Platform Program, 2005DKA21203


Objective To construct and purify the recombinant protein of platelet-activating factor acetylhydrolase (PAF-AH) isoformⅠ, and study the enzyme activity by different substrates. Methods The β subunit of PAF-AH isoformⅠwas cloned and expressed in E. coli. Exogenously expressed recombinant protein was purified to SDS-PAGE homogeneity, and its activity was identified by arylesterase detection. Phenylacetate, l-O-hexadecyl-2-deoxy-2-thioacetyl-sn-glycero-3-phosphocholine (2-Thio PAF) and 1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine (the latter two were commercial plasma PAF-AH substrates) were used for the substrate identification. The plasma type PAF-AH was served as positive control. Results Recombinant protein of β subunit of PAF-AH isoformⅠwas successfully constructed and expressed in E. coli after purification. Compared with positive control, the recombinant protein could hydrolyze phenylacetate and 2-Thio PAF, but could not hydrolyze 1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine. Conclusion Recombinant protein of β subunit of PAF-AH isoformⅠcan be successfully constructed. There are differences in the substrate specification to the two commercial PAF substrates for PAF-AH isoformⅠand plasma type PAF-AH, which provides a quick method to differentiate PAF-AH isoformⅠfrom plasma type PAF-AH.

Key words: platelet-activating factor acetylhydrolase isoformⅠ, plasma platelet-activating factor acetylhydrolase,
recombinant protein,
purification, enzyme activity, substrate

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