Synthetic spider dragline silk proteins and their production in Escherichia coli
Synthetic spider dragline silk proteins and their production in Escherichia coli
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DOI：
10.1007/s002530050883
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发表时间：
1997-01-01
影响因子：
5
通讯作者：
Irwin, SL
中科院分区：
文献类型：
--
作者：
Fahnestock, SR;Irwin, SL
利用人工合成的寡核苷酸，设计合成基因，编码棒纹夜蛾拖丝蛋白1和2的类似物。这些基因是由合成的寡核苷酸组装而成的相对较长(超过300bp)的DNA序列的串联重复序列，并编码高分子量(65-163 kDa)的蛋白质。这两个类似物都能在大肠杆菌中高效生产。较长基因产物的产量和同质性受到合成提前终止的限制，这可能是由于蛋白质合成的过程性错误所致。平均终止率被确定为1100个密码子中有1个到300个密码子中有1个，这取决于基因的长度和同义密码子选择。这两种类似蛋白都可以在没有变性剂的情况下被诱导形成稳定的水溶液。纯化蛋白在稀溶液中的圆二色谱与天然牵引丝在水中的圆二色谱相似，在甲醇和三氟乙醇混合溶剂中的圆二色谱反映了较大的无序结构。
Synthetic genes were designed to encode analogs of the two proteins of Nephila clavipes dragline silk, spidroins 1 and 2. The genes were constructed of tandem repeats of relatively long (more than 300 bp) DNA sequences assembled from synthetic oligonucleotides, and encoded proteins of high molecular mass (65-163 kDa). Both analogs were produced efficiently in Escherichia coli. The yield and homogeneity of the products of longer genes were limited by premature termination of synthesis, probably as a result of processivity errors in protein synthesis. Average termination rates were determined to be 1 in 1100 codons to 1 in 300 codons, depending on the length and synonymous codon choices of the gene. Both analog proteins could be induced to form stable aqueous solutions without denaturants. Circular dichroism spectra of the purified proteins in dilute solution resembled spectra of redissolved natural dragline silk in reflecting a largely disordered structure in water and more ordered structures in mixed solvents with methanol and trifluoroethanol.