Prince J T, McGrath K P, DiGirolamo C M, Kaplan D L
Biotechnology Division, U.S. Army Natick Research, Development, & Engineering Center, Natick, Massachusetts 01760-5020, USA.
Biochemistry. 1995 Aug 29;34(34):10879-85. doi: 10.1021/bi00034a022.
Synthetic genes encoding recombinant spider silk proteins have been constructed, cloned, and expressed. Protein sequences were derived from Nephila clavipes dragline silk proteins and reverse-translated to the corresponding DNA sequences. Codon selection was chosen to maximize expression levels in Escherichia coli. DNA "monomer" sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimers were cloned into a prokaryotic expression vector and the encoded silk proteins were expressed in E. coli upon induction with IPTG. Four multimer, ranging in size from 14.7 to 41.3 kDa, were chosen for detailed analysis. These proteins were isolated by immobilized metal affinity chromatography and purified using reverse-phase HPLC. The composition and identity of the purified proteins were confirmed by amino acid composition analysis, N-terminal sequencing, laser desorption mass spectroscopy, and Western analysis using antibodies reactive to native spider dragline silk. Circular dichroism measurements indicate that the synthetic spider silks have substantial beta-sheet structure.
编码重组蜘蛛丝蛋白的合成基因已被构建、克隆和表达。蛋白质序列源自金黄圆蛛的拖牵丝蛋白，并反向翻译为相应的DNA序列。密码子选择旨在使大肠杆菌中的表达水平最大化。使用“头对尾”构建策略将DNA“单体”序列多聚化，以编码高分子量的合成蜘蛛丝。多聚体被克隆到原核表达载体中，编码的丝蛋白在IPTG诱导下在大肠杆菌中表达。选择了四种大小在14.7至41.3 kDa之间的多聚体进行详细分析。这些蛋白质通过固定化金属亲和色谱法分离，并用反相高效液相色谱法纯化。通过氨基酸组成分析、N端测序、激光解吸质谱和使用对天然蜘蛛拖牵丝有反应的抗体进行的Western分析，确认了纯化蛋白质的组成和身份。圆二色性测量表明，合成蜘蛛丝具有大量的β-折叠结构。
