Fahnestock S R, Irwin S L
Central Science and Engineering Laboratories, E.I. DuPont de Nemours & Co., Wilmington, DE 19880-0328, USA.
Appl Microbiol Biotechnol. 1997 Jan;47(1):23-32. doi: 10.1007/s002530050883.
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.
合成基因被设计用于编码棒络新妇蛛拖丝的两种蛋白质——蜘蛛丝蛋白1和蜘蛛丝蛋白2的类似物。这些基因由从合成寡核苷酸组装而成的相对较长（超过300 bp）的DNA序列串联重复构建而成，并编码高分子量（65 - 163 kDa）的蛋白质。两种类似物均在大肠杆菌中高效产生。较长基因产物的产量和均一性受到合成过早终止的限制，这可能是蛋白质合成过程中持续性错误的结果。根据基因的长度和同义密码子选择，平均终止率确定为每1100个密码子中有1个终止到每300个密码子中有1个终止。两种类似物蛋白在没有变性剂的情况下都能被诱导形成稳定的水溶液。稀溶液中纯化蛋白的圆二色光谱类似于重新溶解的天然拖丝的光谱，反映出在水中结构基本无序，而在含有甲醇和三氟乙醇的混合溶剂中结构更有序。
