嗜热菌Caldicellulosiruptor bescii Athe_0597基因克隆与表达

Cloning and Expression of Athe_0597 Gene from Caldicellulosiruptor bescii

嗜热菌Caldicellulosiruptor bescii能够高效水解木质纤维素,C. bescii分泌的纤维素降解体系中含有多种糖苷水解酶和未知功能的非催化蛋白。未知功能非催化蛋白Athe0597能与多种纤维素底物结合且在纤维素降解体系中高丰度存在。本研究对Athe0597进行了生物信息学分析;以C. bescii基因组为模板,利用PCR技术扩增Athe0597基因;构建pCold I-0597表达质粒并在Escherichia coli BL21 (DE3)中表达,优化诱导表达条件。生物信息学分析显示,Athe0597的前22个氨基酸为信号肽,去除信号肽后的蛋白理论分子质量为54.59 k D,等电点为9.27,亲水性系数和不稳定系数分别为-0.488和23.88,是亲水性稳定蛋白;成功克隆Athe0597基因并在E. coli BL21 (DE3)中成功表达;获得重组蛋白最佳诱导表达条件:诱导剂IPTG浓度为0.25 mmol/L,诱导时间4 h,诱导前菌体浓度OD600nm为0.6,诱导温度30℃。本研究为Athe0597在C. bescii高效水解纤维素中的功能研究提供了理论基础。

Caldicellulosir uptor bescii can efficiently hydrolyze lignocellulose. The cellulose degradation system secreted by C. bescii contains a variety of functional glycoside hydrolases and non-catalytic proteins with unknown functions. Athe0597, a non-catalytic protein of unknown function, has high abundance in the cellulose degradation system and it can be bound to a variety of cellulose substrates. In this study, the bioinformatics analysis on Athe0597 was performed. Using C. bescii genome DNA as the template, Athe0597 gene was amplified by PCR.pCold I-0597 expression vector was constructed and expressed in Escherichia coli BL21(DE3). The expression conditions were optimized. Bioinformatics analysis showed that the first 22 amino acids of Athe0597 were signal peptides, and the theoretical molecular weight of the protein without the signal peptide was 54.59 kD. The isoelectric point was 9.27. The grand average of hydropathicity and the instability coefficient were-0.488 and 23.88, respectively, indicating that Athe0597 was a kind of hydrophilc stable protein. Athe0597 gene was successfully cloned and expressed in E. coli BL21(DE3). IPTG concentration 0.25 mmol/L, induction time 4 h, OD600 nmof the bacteria concentration before induction 0.6 and induction temperature 30 ℃ were proved to be the optimal induction expression conditions. This study provided a theory foundation for functional research of Athe0597 in efficiently hydrolysis of cellulose with C. bescii.