The Cloning, Expression And Modification Study Of The Arginine Deiminase

AimTo synthesize the gene coding for Arginine deiminase (ADI) of Mycoplasma Arginini,to construct prokaryotic expression vector, and to gain engineered recombinant E.coli expressing Arginine deiminase of Mycoplasma Arginini with high capacity; To explore the modification conditions of ADI in conjugation with Polyethylene glycol (PEG) and, to create the separation and purification process of the reaction mixsture; To develop a method to evaluate the activity of PEG-modified ADI, and to check the activity of ADI and PEG-modified ADI.MethodsTo obtain the gene coding for ADI of Mycoplasma Arginini by DNA synthesis and DNA polymerase chain reaction(PCR). To construct prokaryotic expression vector using pBV220 plasmid. To transform the prokaryotic expression plasmid into E.coli DH5a and to select the amp+ clone. To perform the study of fermentation of recombinant E.coli. using 5 liter fermentation system. To modify ADI molecule using PEG(20kD). To explore the key factors that relate to the reaction efficacy, such as temperature, stirring, time and pH etc. To separate and purify the reaction mixture using gel filtration chromatography technology. To examine the molecule weight using Polyacrylamide Gel Electrophoresis(SDS-PAGE). To analyze the purity of recombinant protein by high performance liquid chromatography method, and to study the activity by in vitra arginine degradation method.ResultsThe full length DNA coding for ADI of Mycoplasma Arginini was gained successfully. The prokaryotic expression vector pBV220-ADI was constructed successfully. The recombinant E.coli. expressing ADI of Mycoplasma Arginini was built,and the expression level was above 30% of the whole E.coli. protein. The recombinant ADI was located in the cytoplasm as inclusion body. The fermentation test demonstrated that the engineered E.coli.can growth in large scale with stable expression level. The modification process of ADI with PEG was created successfully. The separation and purification process was successfully established. The single PEG modified ADI can be effectively isolated from the reaction mixture by two times gel filtration chromatography, and the product bulk of 95% purity was gained. The activity quality control method was created successfully by detecting the degradation of arginine in vitro. The activity of ADI and PEG-ADI was 40 IU/mg and 30 IU/mg respectively. The molecule weight of ADI and PEG-ADI was about 45kD and 65kD respectively by SDS-PAGE method. The results of high performance liquid chromatography revealed that the purity of PEG-ADI was above 95%.ConclusionIn this study, the expression of Arginine deiminase was achieved with high capacity via prokaryotic expression system. The resulting protein located in the cytoplasm of recombinant E.coli as inclusion body. Small scale fermentation test reveals that the engineered E.coli can be applied in production. Thus, a stable foundation was established for further research of Arginine deiminase. The optimized reaction condition was created for the modification of ADI in conjugation with PEG. The optimized parameters of the key factors relating to the reaction efficacy were confirmed. Also, we created a simple and effective process to separate and purify the aiming product from reaction mixture and thus, a reliable base for the preparation of PEG-ADI in production scale. Furthermore, the activity quality control method was established successfully by evaluating the degradation quantity of arginine in vitro. To test activity is an effective way in further optimization of the production process. The study provided the potential in treating liver cancer using the new and biologically active PEG-ADI. This study has the importance both in therapeutic theory and in clinical practice.