Abstract
Pyrosequencing is a technology to determine DNA sequence based on extending. The poor stability of luciferase in pyrosequencing reaction mixture leads to disappointed results, which limits the application and promotion of pyrosequencing. In order to set up a heat-stable pyrosequencing system, we used genetic engineering technology to express recombinant thermostable Luciola lateralis luciferase (rt-LlL) with His-tag on N-end. The crude protein was purified with Ni-affinity chromatography. The rt-LlL was obtained, whose molecular mass was about 60 kDa. With commercial Photinus pyralis luciferase (PpL) to be standard, the activity detection demonstrated that the specific activity of rt-LlL was 4.29 × 1010 RLU/mg, which was higher than that of PpL. Another result indicated that rt-LlL kept high activity at 50 °C and more than 90 % of original activity at 40 °C for 25 min. We got correct results and better signals when substituting the commercial PpL with rt-LlL in pyrosequencing system, which laid the foundation of building stable and reliable pyrosequencing system.
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Xu, S., Zou, B., Song, Q., Zhou, G. (2016). Expression of Thermostable Recombinant Luciola lateralis Luciferase and Its Function on Setting Up of Heat-Stable Pyrosequencing System. In: Zhou, G., Song, Q. (eds) Advances and Clinical Practice in Pyrosequencing. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3308-2_15
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DOI: https://doi.org/10.1007/978-1-4939-3308-2_15
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