Pan Hong-Chun, Song Da-Xiang, Zhou Kai-Ya, Zhu Guo-Ping
Laboratory of Molecular Evolution & Biodiversity, College of Life Sciences, Anhui Normal University, Wuhu 241000, China.
Sheng Wu Gong Cheng Xue Bao. 2007 May;23(3):446-51.
RT-PCR was conducted with one degenerate primer designed according to repetitive regions' amino acid sequence of major ampullate spidroin (MaSp) in spiders and adaptor primer in the SMART cDNA Library Construction Kit. By cloning and sequencing of amplified products, one cDNA clone (GenBank Accession No. AY365017) of Argiope amoena MaSp gene was obtained. The deduced amino acid sequence can be distinctly divided into two regions: (1) Repetitive region that consists of an alternating alanine-rich and glycine-rich domain in which many prolines are present; and (2) C-terminal non-repetitive region. The region coding for 272 amino acids of MaSp gene was subcloned into prokaryotic expression vector pET28b(+) and an about 26kD recombinant protein was expressed at high levels in Escherichia coli BL21 (DE3) after induction of IPTG. After being purified with metal-affinity chromatography on Ni(2+) -IDA-Sepharose columns as well as gel filtration chromatography, the recombinant protein was confirmed to be predicted MaSp by means of amino acid composition analysis and N-terminal amino acid sequence analysis. The solubility behavior of recombinant MaSp with C-terminal non-repetitive region in the present study is similar to that of recombinant dragline silk proteins without C-terminal non-repetitive region expressed by bacteria and yeast in the other studies. The result shows that absence or presence of C-terminal non-repetitive region is not a crucial factor affecting the solubility of the recombinant MaSp.
使用根据蜘蛛主要壶腹蛛丝蛋白（MaSp）重复区域的氨基酸序列设计的一条简并引物和SMART cDNA文库构建试剂盒中的接头引物进行RT-PCR。通过对扩增产物进行克隆和测序，获得了一种悦目金蛛MaSp基因的cDNA克隆（GenBank登录号：AY365017）。推导的氨基酸序列可明显分为两个区域：（1）重复区域，由富含丙氨酸和富含甘氨酸的交替结构域组成，其中存在许多脯氨酸；（2）C端非重复区域。将MaSp基因编码272个氨基酸的区域亚克隆到原核表达载体pET28b(+)中，经IPTG诱导后，在大肠杆菌BL21(DE3)中高水平表达了一种约26kD的重组蛋白。经Ni(2+) -IDA-琼脂糖柱金属亲和层析以及凝胶过滤层析纯化后，通过氨基酸组成分析和N端氨基酸序列分析证实该重组蛋白为预测的MaSp。本研究中具有C端非重复区域的重组MaSp的溶解行为与其他研究中细菌和酵母表达的无C端非重复区域的重组拖牵丝蛋白的溶解行为相似。结果表明，C端非重复区域的缺失或存在不是影响重组MaSp溶解性的关键因素。
