Preparation And Characterization Of Sequencing Grade Modified Trypsin & The Experimental Study Of Dorsal Root Ganglion Neurons Primary Culture And Growth In Vitro From Embryonic Rat

Part ⅠObjective:To prepare highly purified, effective, restriction site-specific and chemically stabilized sequencing grade modified trypsin, and apply for protein identification and analysis in proteomics research.Methods:Firstly, raw trypsin isolated from porcine pancreas is reductive methylated with sodium borohydride buffer and formaldehyde following purification by reverse phase chromatography of 15RPC column, producing a stable trypsin that is resistant to tryptic autolysis. Then, the chromatography peak composition of methyl-trypsin was collected. Secondly, methyl-trypsin is further improved by TPCK treatment and is subjected to extensive purification by reverse phase chromatography to remove other non-specific enzyme activity from contaminating proteases. The final product is sequencing grade modified trypsin. Thirdly, the quality of sequencing grade modified trypsin is tested by SDS-PAGE gel electrophoresis, HPLC reversed phase chromatography, catalytic activity on TAME, typical enzyme substrate and characterized by some assays which relate to its application in-gel tryptic digestions following identified by mass spectrometry, peptide spectrum analysis of protein digestion in solution and LTQ mass spectrometry enzyme cleavage site specificity.Results:Purity of sequencing grade modified trypsin is more than 95% in HPLC assay, appears as a single band on SDS-PAGE. The specific activity is 200 TAME units/mgP above. The highly purified and chemically stabilized sequencing grade trypsin gives excellent performance for using in-gel tryptic digestions. And the preparation process of enzyme is stably and repeatable.Conclusion:This process yields a highly purified trypsin product that is effective, highly specific cleavage resulting in a limited number of tryptic peptides and stable, can be widely used in proteomics for peptide mapping and protein identification work at lab. And the stable preparation process can be applied to produce and develop mass spectrum sequencing grade modified trypsin.Part IIObjective:To establish an easy, stable and efficient primary culture and purification systems of isolated dorsal root ganglion (DRG) and DRG neurons derived from embryonic rats in vitro.Methods:DRGs from SD E15 rats were collected, and the explants of DRG were plated and cultured, or the DRGs were dispersed into individual cell suspension with papain dissociation by primary isolated culture methods. The explants and DRG neurons were plated in NeuroBASAL medium containing NGF,5-Fluoro-2’-deoxyuridine and uridine to culture and purify in vitro. Then, the growth regularity of cultured DRG neurons was observed in IncuCyteTM Live-Cell Imaging system. When DRG explants and DRG neurons were cultured for 7 days in vitro, Calcein AM was used for the study of live DRG explants and DRG neurons, and double staining with anti-neuronal nuclei antibody (NeuN) and DAPI to evaluate the purification rate of cultured DRG neurons.Results:Isolated and cultured DRG explants and DRG neurons survived healthily more than a month in vitro in optimized NeuroBASAL medium, and extend the process which formed dense network, the purified rate of DRG neurons could reach to around 90%.Conclusion:The above primary culture systems of DRG explants and DRG neurons in vitro were easy, stable and efficient, can be used as an effective experimental tool and model for the further studies of neuroscience and pharmacology.