Journal of East China Normal University(Natural Sc ›› 2012, Vol. 2012 ›› Issue (4): 67-74.

• Article • Previous Articles     Next Articles

Overexpression and characterization of a bacterial α-N-acetylgalactosaminidase

LIN Yi-gang, WANG Yu-meng,  XIA Gang   

  1. Key Laboratory of Brain Functional Genomics, Ministry of Education, Shanghai Key Laboratory of Brain Functional Genomics, East China Normal University, Shanghai 200062, China
  • Received:2011-05-01 Revised:2011-08-01 Online:2012-07-25 Published:2014-12-15

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

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