Fisher Adam C, DeLisa Matthew P
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, United States of America.
PLoS One. 2008 Jun 11;3(6):e2351. doi: 10.1371/journal.pone.0002351.
Green fluorescent protein (GFP) has undergone a long history of optimization to become one of the most popular proteins in all of cell biology. It is thermally and chemically robust and produces a pronounced fluorescent phenotype when expressed in cells of all types. Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics. Here we report that unlike other well-folded variants of GFP (e.g., GFPmut2), superfolder GFP was spared from elimination when targeted for secretion via the SecYEG translocase. This prompted us to hypothesize that the folding quality control inherent to this secretory pathway could be used as a platform for engineering similar 'superfolded' proteins. To test this, we targeted a combinatorial library of GFPmut2 variants to the SecYEG translocase and isolated several superfolded variants that accumulated in the cytoplasm due to their enhanced folding properties. Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP. Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function. Overall, we demonstrate that laboratory evolution combined with secretory pathway quality control enables sampling of largely unexplored amino-acid sequences for the discovery of artificial, high-performance proteins with properties that are unparalleled in their naturally occurring analogues.
绿色荧光蛋白（GFP）经过了漫长的优化历程，成为细胞生物学中最受欢迎的蛋白质之一。它在热和化学方面都很稳定，在所有类型的细胞中表达时都会产生明显的荧光表型。最近，一种超级折叠GFP被设计出来，具有更高的抗变性能力和更快的折叠动力学。我们在此报告，与GFP的其他折叠良好的变体（如GFPmut2）不同，当通过SecYEG转位酶靶向分泌时，超级折叠GFP不会被清除。这促使我们推测，这种分泌途径固有的折叠质量控制可以用作工程化类似“超级折叠”蛋白质的平台。为了验证这一点，我们将GFPmut2变体的组合文库靶向SecYEG转位酶，并分离出了几种由于折叠特性增强而在细胞质中积累的超级折叠变体。这些GFP变体中的每一个都比亲本GFPmut2蛋白表现出快得多的折叠动力学，其中一种被命名为超快GFP，其折叠速度甚至超过了超级折叠GFP。值得注意的是，相对于GFPmut2，这些GFP变体的比荧光活性几乎没有损失，这表明超级折叠过程可以在不改变蛋白质正常功能的情况下完成。总体而言，我们证明，实验室进化与分泌途径质量控制相结合，能够对大量未探索的氨基酸序列进行采样，以发现具有天然类似物中无与伦比特性的人工高性能蛋白质。
