热稳定生物素化荧光素酶的制备及其在焦测序中的应用

Preparation of Thermostable Biotinlyted Firefly Luciferase and Its Application to Pyrosequencing

新一代大规模焦测序技术需要稳定的可固定化的荧光素酶,为了制备热稳定性好且被生物素化的荧光素酶,本研究采用基因工程方法将生物素羧基载体蛋白C端87个氨基酸残基(BCCP87)与荧光素酶在大肠杆菌E.coli BL21(DE3)中进行融合表达,以实现菌体内直接表达生物素化的荧光素酶(BCCP-LUC);并对北美萤火虫(Photinus pyralis)荧光素酶基因进行了定点突变以增强其热稳定性;用链亲和素包被的磁珠对重组融合蛋白进行固定化,用于焦测序。实验结果表明:突变后的荧光素酶在50℃环境中仍具有活性;在43℃下10min活性保留大于80%,热稳定性明显增强。Western blot分析结果表明,荧光素酶能够在大肠杆菌内生物素化。用链亲和素包被的磁珠结合BCCP-LUC后具有较高活性(2.1×105RLU/μL Beads),经过多次洗涤活性无明显下降。采用微球固定的荧光素酶以及ATP硫酸化酶成功的进行了DNA序列的测定且定量准确,表明固定化的荧光素酶可以应用于焦测序中,为建立高通量大规模芯片焦测序技术提供有效、稳定的工具酶。

The next generation pyrosequencing system requires stable and immobilized luciferase.In order to get stable and immobilized luciferase（LUC） for the large-scale pyrosequencing platform,the genes coding the Photinus pyralis firefly luciferase and 87 C-terminal residues of biotin carboxyl carrier protein（BCCP87） were inserted into plasmid to express biotinylated fussion protein in E.coli BL21（DE3） ,and the gene of luciferase was mutated to get thermostable biotinylated luciferase.The fusion protein was immobilized on streptavidincoated Beads and applied to pyrosequencing.The results of activity detection showed that mutant luciferase had a good tolerance to heat,and still showed activities at 50℃.The mutant LUC remains 80%activities after incubated at 43℃for 10 min.Western blot analysis indicated that the fusion protein was successfully biotinylated in E.coli.The biotinylated luciferase immobilized on magnetic beads coated with streptavidin showed catalytic activity of 2.1×10 5 RLU/μL beads and the activity remained nearly constant even if beadLUC suffered from multiple washing processes.The DNA was accurately and quantitatively sequenced by using luciferase and adenosine triphosphate（ATP） sulfurylase co-immobilized on magnetic beads.This study provides an efficient and thermostable enzyme for the large-scale chip-based pyrosequencing system.