蜘蛛大壶状腺丝蛋白基因的克隆和原核表达

Cloning and Prokaryotic Expression of Major Ampullate Spidroin Gene of Spider

摘要:

以悦目金蛛（Argiope amoena）丝腺SMART RACE cDNA文库为模板进行RT-PCR，克隆了1条大壶状腺丝蛋白（major ampullate spidroin, MaSp）基因cDNA序列。该条cDNA序列编码的氨基酸序列可区分为两部分：(1)富含丙氨酸的片段和富含甘氨酸的片段相间排列构成的重复氨基酸序列区，并且富含甘氨酸的片段中有脯氨酸分布；（2）约100个氨基酸残基组成的C末端非重复氨基酸序列区。把MaSp基因cDNA序列亚克隆到质粒pET28b(+)中，构建原核表达质粒pET28b(+)-MaSp，表达质粒转化大肠杆菌BL21(DE3)，用IPTG诱导表达。SDS-PAGE、氨基酸组成测定和N末端氨基酸序列测定的结果表明，表达产物为重组MaSp，表达量约为40mg/L。还对C末端非重复氨基酸序列对重组MaSp在水媒介中溶解性的影响进行了探讨。

Abstract:

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^TM 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 Ni²⁺-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.