Direct evolution of GlcNAc 2-epimerase by DNA shuffling

• Main Author: 林芝安
• Other Authors: 洪純隆
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/64078694293094964521

碩士 === 高雄醫學大學 === 醫學研究所 === 89 === PART IIn mammalian system, NeuAc is known to be biosynthesized either from UDP-N-acetyl-D-glucosamine by an action of UDP-N-acetyl-D-glucosamine 2-epimerase or from N-acetyl-D-glucosamine by N-acetyl-D-glucosamine 2-epimerase ( GlcNAc 2-epimerase ). However, the physiological function of the GlcNAc 2-epimerase in NeuAc biosynthesis has not been fully evaluated. To clarify the role of GlcNAc 2-epimerase in NeuAc biosynthesis the enzyme and its gene were isolated from porcine kidney cortex. Escherichia coli cells transformed with the gene expressed the GlcNAc 2-epimerase having the same properties as those of the GlcNAc 2-epimerase from porcine kidney. Homology search for the cloned gene revealed that the GlcNAc 2-epimerase was identical with renin-binding protein ( RnBP ) in porcine kidney ( Inoue, H., Fukui, K., Takahashi, S., and Miyake, Y.1990 J.Biol.Chem. 265, 6556-6561 ) ( identity:99.6%in nucleotide sequence, 99% in amino acid sequence ). Recently, N-Acetylneuraminate lyase ( aldolase ) and N-acyl-D- glucosamine 2-epimerase had been cloned and overexpressed in Escherichia coli. Simultaneous use of these two enzymes and feeding of appropriate amounts of pyruvate to the reaction to mixture made possible the high conversion of Neu5Ac from GlcNAc. However, GlcNAc 2-epimerase required ATP for activation. The cost of ATP is not applicable for industrial use. It is required to reengineer and search new source of GlcNAc 2-epimerase which exhibits no ATP requirement. We have used reverse transcriptase polymerase chain reaction ( RT-PCR ) method to amplify cDNA from three species of human, rat and porcine. Then to subclone and recombinant the PCR products to pET-14b and transform to E.coli to overexpress N-acyl-D-glucosamine 2-epimerase protein. The enzyme activity maintain a status. By the method of “the incremental truncation for the creation of hybrid enzymes”, we were used the internal NcoI cutting site to obtaine several clones, and the recombinant enzyme activity is shown increase 5~10%. In the future, we will try to engineer these three GlcNAc 2-epimerase proteins by DNA shuffling combined GFPuv-fusion to find the more activity of the recombinant GlcNAc 2-epimerase than the wild type GlcNAc 2-epimerase for the highthroughput method by Industrial Technology Research Institute. Attempt to gain ATP-independent and low Km value GlcNAc 2-epimerase. PART II Autoantibodies to intracellular antigen are a hallmark of autoimmune diseases, although their role in disease pathogenesis is unclear. Centrosomes are organelles involved in the organization of the mitotic spindle and they are targets of autoantibodies in autoimmune diseases. We used recombinant centrosome autoantigens, centrosome-specific antibodies, and immunoassays to demonstrate that a significant proportion of autoimmune patients exhibited centrosome reactivity. One centrosome protein cloned in our laboratory were used to screen 73 autoimmune sera by Western blotting. The same sera were screened by immunofluorescence using centrosome-specific antibodies to distinguish centrosomes from nuclear speckles commonly stained by autoimmune diseases. Using these criteria, 25-45 % of autoimmune patients were autoreactive to centrosomes, a larger percentage than reacted with all other known autoimmune autoantigens. Only small percentage of normal individuals had centrosome reactivity. This results demonstrate that centrosome autoantibodies are a major component of autoreactivity in autoimmune diseases. Centrosome autoantigen may be useful in studying the development of autoantibodies and chronic inflammation in autoimmune diseases.