›› 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

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Abstract:

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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