Construction, purification and substrate specificity identification of recombinant human platelet-activating factor acetylhydrolase isoformⅠ / 上海交通大学学报（医学版）

ABSTRACT

Objective To construct and purify the recombinant protein of platelet-activating factor acetylhydrolase (PAF-AH) isoform I , and study the enzyme activity by different substrates. Methods The (3 subunit of PAF-AH isoform I was 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, 1-O-hexadecyl-2-deoxy-2-thioacetyl-sn-glycero-3-phosphocholine ( 2-Thio PAF) and l-myristoy1-2-( 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 isoform I was 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 l-myristoyl-2-( 4-nitrophenylsuccinyl) phosphatidylcholine. Conclusion Recombinant protein of β subunit of PAF-AH isoform I can be successfully constructed. There are differences in the substrate specification to the two commercial PAF substrates for PAF-AH isoform I and plasma type PAF-AH, which provides a quick method to differentiate PAF-AH isoform I from plasma type PAF-AH.