EXPRESSION OF THERMOSTABLE RECOMBIANT LUaOLALATERALIS LUCIFERASE AND APPLICATION PYROSEQUENCING SYSTEM

摘要

Pyrosequencing is a widely-used tool for DNA sequence analysis. However, the instability of pyrosequencing reagents caused by fragile luciferase usually leads to unsatisfied results, which limits the application of pyrosequencing. In order to improve the stability of pyrosequencing reagents, we expressed recombinant thermostable Luciola lateralis Lucif erase (rt-L1L) with a His-tag on its N-terminus by genetic engineering technology. The purified rt-LlL with the molecular mass of 60 KDa was obtained by Ni-affinity chromatography. Using commercial Photinus pyralis lucif erase as a standard, the specific activity of rt-LlL was determined as 4. 29 × 1 010 RLU/mg, which was higher than that of commercial Photinus pyralis luciferase. Moreover, the thermostability of rt-LlL was investigated, and the results showed that rt-LlL had activity at 50℃, and remained 90% of activity after incubated at 40℃ for 25 min. Finally, rt-LlL was used to substitute commercial Photinus pyralis luciferase in conventional pyrosequencing reagent to get thermostable pyrosequencing reagent, whose activity would not lose when incubated at 37℃ for 1 h, while the conventional pyrosequencing reagent showed little activity when incubated at 37℃ for 1 h. The results indicated that the thermostable pyrosequencing reagent was more stable than conventional pyrosequencing reagent, and was better on application in biological field.