家蚕蛋白酪氨酸磷酸酶基因克隆、表达纯化与结构分析

Cloning, expression, purification and structure analysis of protein tyrosine phosphatase of Bombyx mori

摘要:

蛋白酪氨酸磷酸酶 (Protein tyrosine phosphatase, PTP, EC 3.1.3.48) 特异性地催化去除磷酸化修饰的酪氨酸残基上的磷酸基团，导致蛋白去磷酸化，从而调控了细胞生长、增殖、分化和免疫等生命活动。家蚕Bombyx mori蛋白酪氨酸磷酸酶h (BmPTP-h) 参与了核型多角体病毒 (Nucleopolyhedrovirus，NPV) 在家蚕体内的复制过程，但目前对于BmPTP-h结构和性质的了解并不多。本文从家蚕中肠克隆了BmPTP-h基因编码序列，分析了BmPTP-h的基因组结构、mRNA结构、序列特征、二级结构和溶液中的状态。同源氨基酸序列比对分析表明BmPTP-h与多种昆虫NPV的PTP序列具有高相似度，暗示了它们可能具有共同的起源和相似的功能。文中构建了原核表达载体，通过大肠杆菌在25 ℃下表达获得了可溶性的重组BmPTP-h，利用Ni-NTA亲和层析纯化了BmPTP-h。凝胶过滤分析显示BmPTP-h在溶液中可以形成聚集体和单体。圆二色光谱分析显示重组的BmPTP-h包含α螺旋结构，升高温度导致BmPTP-h的α螺旋结构去折叠，α螺旋结构含量下降。这些研究为深入研究BmPTP-h的结构和调控机理提供了基础。

Abstract:

Protein tyrosine phosphatase (PTP, EC 3.1.3.48) specifically catalyzes the removal of phosphate groups from phosphorylated tyrosine residues, resulting in protein dephosphorylation, thus regulates life activities such as cell growth, proliferation, differentiation and immunization. Protein tyrosine phosphatase h of Bombyx mori (BmPTP-h) is involved in the replication of nucleopolyhedrovirus (NPV) in Bombyx mori, but the structure and properties of BmPTP-h are little known so far. In this study, the coding sequence of BmPTP-h gene was cloned from the midgut of Bombyx mori, and its genomic structure, mRNA structure, sequence signature, secondary structure and the state in solution were analyzed. Homologous amino acid sequences alignment analysis indicated that BmPTP-h had a high similarity to PTP sequences of numbers of insect NPVs, implying that they may have a common ancestor and similar function. We constructed a prokaryotic expression vector, expressed and obtained the soluble recombinant BmPTP-h in Escherichia coli at 25 ℃, and purified BmPTP-h using Ni-NTA affinity chromatography. Gel filtration analysis showed that BmPTP-h was able to form aggregate and monomer in solution. Circular dichroism spectroscopy analysis showed that the recombinant BmPTP-h contained α-helix structure. Increasing temperature resulted in the unfolding of the α-helix structure of BmPTP-h and the decrease of the α-helix structure content of BmPTP-h. These studies provide a basis to better study the structure and regulation mechanism of BmPTP-h.