Crameri A, Whitehorn E A, Tate E, Stemmer W P
Affymax Research Institute, Palo Alto, CA 94304, USA.
Nat Biotechnol. 1996 Mar;14(3):315-9. doi: 10.1038/nbt0396-315.
Green fluorescent protein (GFP) has rapidly become a widely used reporter of gene regulation. However, for many organisms, particularly eukaryotes, a stronger whole cell fluorescence signal is desirable. We constructed a synthetic GFP gene with improved codon usage and performed recursive cycles of DNA shuffling followed by screening for the brightest E. coli colonies. A visual screen using UV light, rather than FACS selection, was used to avoid red-shifting the excitation maximum. After 3 cycles of DNA shuffling, a mutant was obtained with a whole cell fluorescence signal that was 45-fold greater than a standard, the commercially available Clontech plasmid pGFP. The expression level in E. coli was unaltered at about 75% of total protein. The emission and excitation maxima were also unchanged. Whereas in E. coli most of the wildtype GFP ends up in inclusion bodies, unable to activate its chromophore, most of the mutant protein is soluble and active. Three amino acid mutations appear to guide the mutant protein into the native folding pathway rather than toward aggregation. Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled GFP mutant showed a 42-fold improvement over wildtype GFP sequence, and is easily detected with UV light in a wide range of assays. The results demonstrate how molecular evolution can solve a complex practical problem without needing to first identify which process is limiting. DNA shuffling can be combined with screening of a moderate number of mutants. We envision that the combination of DNA shuffling and high throughput screening will be a powerful tool for the optimization of many commercially important enzymes for which selections do not exist.
绿色荧光蛋白（GFP）已迅速成为广泛应用的基因调控报告分子。然而，对于许多生物体，尤其是真核生物而言，更强烈的全细胞荧光信号是很有必要的。我们构建了一个密码子使用优化的合成GFP基因，并进行了DNA改组的递归循环，随后筛选出最亮的大肠杆菌菌落。使用紫外光进行视觉筛选，而非荧光激活细胞分选（FACS）选择，以避免激发最大值发生红移。经过三轮DNA改组后，获得了一个突变体，其全细胞荧光信号比市售的Clontech质粒pGFP（一种标准品）强45倍。在大肠杆菌中的表达水平未变，约占总蛋白的75%。发射和激发最大值也未改变。在大肠杆菌中，大多数野生型GFP最终形成包涵体，无法激活其发色团，而大多数突变蛋白是可溶且有活性的。三个氨基酸突变似乎引导突变蛋白进入天然折叠途径而非聚集途径。在中华仓鼠卵巢（CHO）细胞中表达时，这种改组后的GFP突变体比野生型GFP序列表现出42倍的提升，并且在广泛的检测中用紫外光很容易检测到。结果表明分子进化如何能够在无需首先确定哪个过程是限制因素的情况下解决复杂的实际问题。DNA改组可以与适度数量突变体的筛选相结合。我们设想DNA改组和高通量筛选的结合将成为优化许多不存在选择方法的重要商业酶的有力工具。
