Kahn Jason S, Ruiz Roanna C H, Sureka Swati, Peng Songming, Derrien Thomas L, An Duo, Luo Dan
Department of Biological and Environmental Engineering, Cornell University , Ithaca, New York 14853, United States.
Department of Biomedical Engineering, Cornell University , Ithaca, New York 14853, United States.
Biomacromolecules. 2016 Jun 13;17(6):2019-26. doi: 10.1021/acs.biomac.6b00183. Epub 2016 May 17.
Protein expression and selection is an essential process in the modification of biological products. Expressed proteins are selected based on desired traits (phenotypes) from diverse gene libraries (genotypes), whose size may be limited due to the difficulties inherent in diverse cell preparation. In addition, not all genes can be expressed in cells, and linking genotype with phenotype further presents a great challenge in protein engineering. We present a DNA gel-based platform that demonstrates the versatility of two DNA microgel formats to address fundamental challenges of protein engineering, including high protein yield, isolation of gene sets, and protein display. We utilize microgels to show successful protein production and capture of a model protein, green fluorescent protein (GFP), which is further used to demonstrate a successful gene enrichment through fluorescence-activated cell sorting (FACS) of a mixed population of microgels containing the GFP gene. Through psoralen cross-linking of the hydrogels, we have synthesized DNA microgels capable of surviving denaturing conditions while still possessing the ability to produce protein. Lastly, we demonstrate a method of producing extremely high local gene concentrations of up to 32 000 gene repeats in hydrogels 1 to 2 μm in diameter. These DNA gels can serve as a novel cell-free platform for integrated protein expression and display, which can be applied toward more powerful, scalable protein engineering and cell-free synthetic biology with no physiological boundaries and limitations.
蛋白质表达与筛选是生物制品改造过程中的一个关键环节。从多样的基因文库（基因型）中，根据期望的特性（表型）筛选出表达的蛋白质，由于多样细胞制备过程中固有的困难，基因文库的规模可能受到限制。此外，并非所有基因都能在细胞中表达，而且将基因型与表型联系起来在蛋白质工程中仍是一个巨大挑战。我们展示了一个基于DNA凝胶的平台，该平台展现了两种DNA微凝胶形式的多功能性，以应对蛋白质工程的基本挑战，包括高蛋白产量、基因集分离以及蛋白质展示。我们利用微凝胶成功实现了蛋白质生产并捕获了一种模型蛋白——绿色荧光蛋白（GFP），该蛋白进一步用于通过对含有GFP基因的混合微凝胶群体进行荧光激活细胞分选（FACS）来证明成功的基因富集。通过水凝胶的补骨脂素交联，我们合成了能够在变性条件下存活同时仍具备生产蛋白质能力的DNA微凝胶。最后，我们展示了一种在直径为1至2μm的水凝胶中产生高达32000个基因重复的极高局部基因浓度的方法。这些DNA凝胶可作为一个用于整合蛋白质表达与展示的新型无细胞平台，可应用于更强大、可扩展的蛋白质工程以及无生理界限和限制的无细胞合成生物学。
