家蚕bHLH转录因子Bmsage可溶性表达、纯化与结构分析
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国家重点基础研究发展计划 (973计划) (No. 2012CB114602),国家自然科学基金 (Nos. 31402139, 31572465),重庆市基础科学与前沿技术研究专项 (Nos. cstc2015jcyjA00040, cstc2015jcyjBX0035),中央高校基本科研业务费 (No. XDJK2013A019),西南大学博士基金 (No. SWU112111) 资助。


Soluble expression, purification and structural analysis of the bHLH transcription factor Bmsage of Bombyx mori
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National Basic Research Program of China (973 Program) (No. 2012CB114602), National Natural Science Foundation of China (Nos. 31402139, 31572465), Chongqing Research Program of Basic Research and Frontier Technology (Nos. cstc2015jcyjA00040, cstc2015jcyjBX0035), Fundamental Research Funds for the Central Universities (No. XDJK2013A019), Start-up Grant from Southwest University (No. SWU112111).

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    摘要:

    碱性螺旋-环-螺旋 (Basic helix-loop-helix, bHLH) 转录因子在生命活动过程中发挥着重要的调控作用。Bmsage是家蚕丝腺中高量表达的一类bHLH转录因子,不仅参与了丝腺细胞在胚胎期的发育调控,而且对蚕丝蛋白的合成也有至关重要的调控作用,然而当前对其性质和结构了解不多。为研究其性质、结构和生物学功能,构建了NusA、MBP、SUMO、Trx和His融合标签的Bmsage重组原核表达载体,通过改变诱导温度和IPTG浓度,确定了Bmsage在大肠杆菌中可溶性表达的最佳载体和表达条件,进而通过镍柱亲和层析纯化获得了Bmsage,利用圆二色光谱研究了其二级结构。结果表明,NusA与MBP标签可显著增强Bmsage在大肠杆菌中的可溶性表达,但难以与Bmsage分离。SUMO标签有一定的助溶效果,并且能够与Bmsage有效分离。其他标签对Bmsage可溶性表达的效果不明显。圆二色光谱分析表明Bmsage含有α-helix结构,推测SUMO标签可能促进了Bmsage折叠形成类天然的结构。这些工作为深入研究Bmsage的性质、结构与功能建立了基础,同时也为其他类似蛋白的表达纯化提供了参考。

    Abstract:

    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.

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何华伟,位曙光,王叶菁,刘莉娜,李珍珍,赵朋,常怀普,赵萍. 家蚕bHLH转录因子Bmsage可溶性表达、纯化与结构分析[J]. 生物工程学报, 2016, 32(10): 1395-1407

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  • 收稿日期:2016-03-21
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  • 在线发布日期: 2016-09-23
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