Efficient Preparation Of Glyceraldehyde3-Phosphate Dehydrogenase And The Applications In The Prokaryotic And Eukaryotic

Glyceraldehyde3-phosphate dehydrogenase (GAPDH) is a key enzyme of glycolysisreactions to provide energy for a variety of life activities of organisms. The GAPDH gene isknown as a housekeeping gene and expressed highly in tissues and cells, under normalcircumstances, It is often used as the internal reference of the RT-PCR, Westernt Blotexperiments.The prokaryotic expression system was selected in our topics to efficiently express ofglyceraldehyde3-phosphate dehydrogenase in rat PC12cells.We selected the bothTAT-pET28b-GAPDH and pET28b-GAPDH. The results showed that we efficiently preparedglyceraldehydes3-phosphate dehydrogenase and have an important enzyme activity in vitro.In addition, we identified the endogenous glyceraldehyde3-phosphate dehydrogenation inprokaryotes (E. coli different strains HB101,OP50, BL21, DH5α), eukaryotic (yeast,Caenorhabditis elegans, rat, mouse) on the normal culture conditions and different treatmentconditions (different in concentration of inducer and processing time, different concentrationsof Paraquat and sulphite and processing time). The results showed that the expression ofglyceraldehyde3-phosphate dehydrogenase was not affected and remained relatively stable.So in prokaryotic and eukaryotic glyceraldehyde3-phosphate dehydrogenase can be used asinternal standard protein.In summary, this study not only improved the traditional prokaryotic expressiontechnology of preparation of glyceraldehydes3-phosphate, to achieve its high efficientpreparation and an important enzyme activity in vitro, but also to explore its initial animportant application as a standard of prokaryotic and eukaryotic proteins quantitativeresearch.