为了研究OsLEA5 c蛋白对其他蛋白的保护效应,在大肠杆菌DL21中异源表达OsLEA5 c蛋白,采用HiTrapTM chelating HP层析柱纯化OsLEA5c蛋白。纯化的OsLEA5c蛋白用于高温、脱水和冻融胁迫下对乳酸脱氢酶的保护效应研究。结果表明,在 PBS 缓冲体系中,50℃处理10、20、30 min后,乳酸脱氢酶的活性分别残留43.20%、24.53%、17.55%；而按1∶1的质量比加入纯化的OsLEA5 c蛋白后,乳酸脱氢酶活性分别保留85.77%、74.24%、65.27%。干燥脱水5 h后,乳酸脱氢酶活性仅保留2.55%~4.19%；按2∶1、5∶1和10∶1的质量比加入OsLEA5 c蛋白,乳酸脱氢酶活性分别保留17.59%、24.73%、36.39%。反复冻融5次,乳酸脱氢酶活性残留9.41%~12.14%；而按2∶1、5∶1和10∶1的质量比加入纯化的OsLEA5 c 蛋白,乳酸脱氢酶活性分别保留28.09%、69.44%、95.16%。表明OsLEA5 c蛋白对其他蛋白质具有保护作用,能减少高温、脱水和冻融胁迫对蛋白质的损伤。%In order to investigate protective effect of OsLEA5c protein on other proteins,OsLEA5c protein was heterologously expressed in Escherichia coli DL21,and purified using HiTrapTM chelating HP column. The purified OsLEA5 c protein was used to study the protective effect of the OsLEA5 c protein on lactate dehydrogenase( LDH) activity under heating,drying,and freeze-thaw stresses. LDH was incubated in PBS buffer at 50 ℃for 10,20,30 min,its activity was decreased to 43. 20%,24. 53% and 17. 55%,respec-tively. However,when OsLEA5c protein was added into LDH solution at a mass ratio of 1∶1 (OsLEA5c∶LDH),enzyme activity remained at 85. 77%,74. 24% and 65. 27%,respectively,after treatment in the same way. After air-drying for 5 h,the residual activity of LDH were 2. 55%—4. 19%; while OsLEA5c was added into LDH solution at mass ratios of 2∶1,5∶1 and 10∶1,its residual activity were 17. 59%, 24. 73% and 36. 39%,respectively. After 5 cycles of freeze-thaw treatments,the activity of residual LDH was decreased to 9. 41%—12. 14%,while OsLEA5c was added to LDH solution at mass ratios of 2∶1, 5∶1 and 10∶1,its residual activity were 28. 09%,69. 44% and 95. 16 %,respectively,after freeze-thaw treatments. Therefore,the result suggested that OsLEA5c protein could protect other protein and decrease damage from heating,drying,and freeze-thaw stresses.
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