He Huawei, Wei Shuguang, Wang Yejing, Liu Lina, Li Zhenzhen, Zhao Peng, Chang Huaipu, Zhao Ping
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, China.
Chongqing Engineering and Technology Research Center for Novel Silk Materials, College of Biotechnology, Southwest University, Chongqing 400715, China.
Sheng Wu Gong Cheng Xue Bao. 2016 Oct 25;32(10):1395-1407. doi: 10.13345/j.cjb.160110.
Basic helix loop helix (bHLH) transcription factor plays an important role in biological processes. Bmsage is a class of bHLH transcription factor highly expressed in the silk gland of Bombyx mori, which is not only involved in the developmental regulation of the silk gland cells at the embryonic period, but also plays a crucial regulatory role during the synthesis of silk protein. However, currently, much of the property and structure of Bmsage is still remained unknown. To study the property, structure and biological role of Bmsage, we constructed several prokaryotic expression vectors of Bmsage fused with NusA, MBP, SUMO, Trx and His tags, respectively, then screened and determined the best soluble expression vector and condition of Bmsage in Escherichia coli combining with the induction temperature and IPTG concentration, and further purified the recombinant Bmsage by Ni-column affinity chromatography according to the established expression condition and characterized its secondary structure using circular dichroism spectra. The results showed that NusA and MBP could significantly enhance the soluble expression of Bmsage in E. coli, but it was difficult to separate Bmsage from these tags. SUMO could not only increase the soluble expression of Bmsage in E. coli to a certain degree, but also be effectively separated from Bmsage. Other tags did not effectively promote the soluble expression of Bmsage in E. coli. Circular dichroism spectra showed that the purified Bmsage had well-defined α-helix structure in solution, indicating that SUMO may promote the correct folding of Bmsage into native-like structure. These work not only establish a foundation for further study of the property, structure and function of Bmsage, but also provide a reference for the expression and purification of other similar proteins.
碱性螺旋-环-螺旋（bHLH）转录因子在生物过程中发挥着重要作用。Bmsage是一类在家蚕丝腺中高度表达的bHLH转录因子，它不仅参与胚胎期丝腺细胞的发育调控，而且在丝蛋白合成过程中也起着关键的调控作用。然而，目前Bmsage的许多性质和结构仍然未知。为了研究Bmsage的性质、结构和生物学作用，我们分别构建了几种与NusA、MBP、SUMO、Trx和His标签融合的Bmsage原核表达载体，然后结合诱导温度和IPTG浓度，筛选并确定了Bmsage在大肠杆菌中的最佳可溶性表达载体和条件，并根据既定的表达条件通过镍柱亲和层析进一步纯化重组Bmsage，利用圆二色光谱对其二级结构进行了表征。结果表明，NusA和MBP能显著增强Bmsage在大肠杆菌中的可溶性表达，但难以将Bmsage与这些标签分离。SUMO不仅能在一定程度上提高Bmsage在大肠杆菌中的可溶性表达，而且能有效地与Bmsage分离。其他标签不能有效地促进Bmsage在大肠杆菌中的可溶性表达。圆二色光谱表明，纯化后的Bmsage在溶液中具有明确的α-螺旋结构，表明SUMO可能促进Bmsage正确折叠成类似天然的结构。这些工作不仅为进一步研究Bmsage的性质、结构和功能奠定了基础，也为其他类似蛋白质的表达和纯化提供了参考。
