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| Optimization of conditions for preparation of recombinant human CYP2C9 yeast microsomes by glass bead crushing method |
| WU Yue-zhang1 Zeinab Abdelrahaman1 QIAN Wen2 Ikeda Katsuya3 YANG Chang-qing1,2▲ |
1.College of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Jiangsu Province, Nanjing 211198, China;
2.Bei Rui-si Biomedical Science and Technology Company Limited of Nanjing, Jiangsu Province,Nanjing 211100, China;
3.Fujikawa & Co.Ltd, Tokyo 103-0021, Japan |
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Abstract Objective To prepare the most active recombinant human CYP2C9 yeast microsomes by optimizing the conditions of glass bead crushing method, thus provide and establish more sensitive and rapid in vitro metabolic screening model with the minimum metabolic content degradation.Methods Recombinant CYP2C9 human microsomes content was prepared by glass bead crushing method.By changing the number of vortex oscillations and the amount of glass beads, the conditions of preparing microspheres were optimized.Lowry method was used to determine protein and metabolite concentration in microsomes.The reaction with microsomes was carried out using Diclofenac, which was a typical substrate for 2C9 microsomes.The protein activity of microsomes was determined by the yield of diclofenac metabolite (4-hydroxy diclofenac) corresponding to unit protein (mg/ml) per unit time (h).Results With the increase of the number of vortex oscillations and the amount of glass beads, the content of the prepared microsomal protein increased.When the number of vortices oscillated 30 times (30 s per vortex oscillation, 60 s of cooling) and the amount of glass beads was 0.75 g, the metabolite production and the corresponding metabolite production per unit of protein per unit of time were the largest, 1.21 μg and 0.06 μg/[(mg/ml)·h]respectively.Conclusion The optimal condition for preparation of recombinant human CYP2C9 yeast microsomes with minimum content degradation using glass beads lysis process was 30 cycle consist of (30 s per vortex oscillation, 60 s of cooling) and amount of 0.75 g glass beads.
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