Department of Biological Sciences & Molecular Biosensor and Imaging Center, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA.
Mol Biotechnol. 2013 Jul;54(3):829-41. doi: 10.1007/s12033-012-9631-7.
Directed evolution is an exceptionally powerful tool that uses random mutant library generation and screening techniques to engineer or optimize functions of proteins. One class of proteins for which this process is particularly effective is antibodies, where properties such as antigen specificity and affinity can be selected to yield molecules with improved efficacy as molecular labels or in potential therapeutics. Typical antibody structure includes disulfide bonds that are required for stability and proper folding of the domains. However, these bonds are unable to form in the reducing environment of the cytoplasm, stymieing the effectiveness of optimized antibodies in many research applications. We have removed disulfide-forming cysteine residues in a single chain antibody fluorogen-activating protein (FAP), HL4, and employed directed evolution to select a derivative that is capable of activity in the cytoplasm. A subsequent round of directed evolution was targeted at increasing the overall brightness of the fluoromodule (FAP-fluorogen complex). Ultimately, this approach produced a novel FAP that exhibits strong activation of its cognate fluorogen in the reducing environment of the cytoplasm, significantly expanding the range of applications for which fluoromodule technology can be utilized.
定向进化是一种非常强大的工具，它利用随机突变文库的生成和筛选技术来设计或优化蛋白质的功能。在一类特别适合这种方法的蛋白质中，抗体就是其中之一，通过这个过程可以选择具有改善的效力的分子作为分子标记或在潜在的治疗方法中。抗体的典型结构包括二硫键，这些键对于结构域的稳定性和正确折叠是必需的。然而，这些键在细胞质的还原环境中无法形成，这限制了优化后的抗体在许多研究应用中的有效性。我们已经去除了单链抗体荧光素酶激活蛋白 (FAP) HL4 中的形成二硫键的半胱氨酸残基，并利用定向进化选择了一种能够在细胞质中发挥作用的衍生物。随后的一轮定向进化旨在提高荧光模块（FAP-荧光素酶复合物）的整体亮度。最终，这种方法产生了一种新型的 FAP，它在细胞质的还原环境中强烈激活其同源荧光素酶，显著扩展了荧光模块技术可应用的范围。
