Heterogeneous Biocatalysis Laboratory, CICbiomaGUNE, Paseo Miramón 182. Edificio empresarial "C", 20014 San Sebastián, Spain.
Heterogeneous Biocatalysis Laboratory, Instituto de Síntesis Química y Catálisis Homogénea (iSQCH), CSIC-Universidad de Zaragoza, C/Pedro Cerbuna 12, 50009 Zaragoza, Spain.
ACS Synth Biol. 2020 Nov 20;9(11):2971-2978. doi: 10.1021/acssynbio.0c00462. Epub 2020 Nov 10.
The rapid demand for protein-based molecules has stimulated much research on cell-free protein synthesis (CFPS); however, there are still many challenges in terms of cost-efficiency, process intensification, and sustainability. Herein, we describe the microcompartmentalization of CFPS of superfolded green fluorescent protein (sGFP) in alginate hydrogels, which were casted into a μ-channel device. CFPS was optimized for the microcompartmentalized environment and characterized in terms of synthesis yield. To extend the scope of this technology, the use of other biocompatible materials (collagen, laponite, and agarose) was explored. In addition, the diffusion of sGFP from the hydrogel microenvironment to the bulk was demonstrated, opening a promising opportunity for concurrent synthesis and delivery of proteins. Finally, we provide an application for this system: the CFPS of enzymes. The present design of the hydrogel μ-channel device may enhance the potential application of microcompartmentalized CFPS in biosensing, bioprototyping, and therapeutic development.
对基于蛋白质的分子的快速需求刺激了大量无细胞蛋白质合成 (CFPS) 的研究；然而，在成本效益、过程强化和可持续性方面仍然存在许多挑战。在此，我们描述了超折叠绿色荧光蛋白 (sGFP) 的 CFPS 在藻酸盐水凝胶中的微区化，藻酸盐水凝胶被浇铸到 μ 通道装置中。针对微区化环境对 CFPS 进行了优化，并根据合成产率进行了表征。为了扩展该技术的范围，探索了使用其他生物相容性材料（胶原、拉蓬土和琼脂糖）的方法。此外，还证明了 sGFP 从水凝胶微环境扩散到本体，为蛋白质的同时合成和输送开辟了广阔的前景。最后，我们提供了该系统的一个应用：酶的 CFPS。水凝胶 μ 通道装置的当前设计可能会增强微区化 CFPS 在生物传感、生物原型制作和治疗开发中的潜在应用。
