Abstract: The coding sequence of alpha-N-acetylgalactosaminidase (A4) was amplified from genomic DNA of Chryseobacterium meningosepticum and subcloned into pET24a, which was then transformed into BL21(DE3) for overexpression of His-A4. The overexpressed His-A4 enzyme was purified by using affinity chromatography and its activity was comparable to that previously reported by using a conventional method with an artificial substrate. To better measure the activity of α-N-acetylgalactosaminidase in real application, we established a novel method in which we directly used the surface antigen of red blood cell as substrate and applied ELISA to the detection of un-cleaved antigen. The activity of His-A4 was evaluated in the new ELISA method and was demonstrated to be able to decrease the blood cell surface antigen-antibody reaction in concentration- and time-dependent manner.

Key words: α-N-acetylgalactosaminidase, recombination protein, protein purification, ELISA method