Ibáñez-Fonseca Arturo, Alonso Matilde, Arias Francisco Javier, Rodríguez-Cabello José Carlos
BIOFORGE Lab, University of Valladolid - CIBER-BBN , Paseo de Belén 19, 47011 Valladolid, Spain.
Bioconjug Chem. 2017 Mar 15;28(3):828-835. doi: 10.1021/acs.bioconjchem.6b00738. Epub 2017 Feb 15.
In the last decades, recombinant structural proteins have become very promising in addressing different issues such as the lack of traceability of biomedical devices or the design of more sensitive biosensors. Among them, we find elastin-like recombinamers (ELRs), which can be designed to self-assemble into diverse structures, such as hydrogels. Furthermore, they might be combined with other protein polymers, such as silk, to give silk-elastin-like recombinamers (SELRs), holding the properties of both proteins. In this work, due to their recombinant nature, we have fused two different fluorescent proteins (FPs), i.e., the green Aequorea coerulescens enhanced green fluorescent protein and the near-infrared eqFP650, to a SELR able to form irreversible hydrogels through physical cross-linking. These recombinamers showed an emission of fluorescence similar to the single FPs, and they were capable of forming hydrogels with different stiffness (G' = 60-4000 Pa) by varying the concentration of the SELR-FPs. Moreover, the absorption spectrum of SELR-eqFP650 showed a peak greatly overlapping the emission spectrum of the SELR-Aequorea coerulescens enhanced green fluorescent protein. Hence, this enables Förster resonance energy transfer (FRET) upon the interaction between two SELR molecules, each one containing a different FP, due to the stacking of silk domains at any temperature and to the aggregation of elastin-like blocks above the transition temperature. This effect was studied by different methods, and a FRET efficiency of 0.06-0.2 was observed, depending on the technique used for its calculation. Therefore, innovative biological applications arise from the combination of SELRs with FPs, such as enhancing the traceability of hydrogels based on SELRs intended for tissue engineering, the development of biosensors, and the prediction of FRET efficiencies of novel FRET pairs.
在过去几十年中，重组结构蛋白在解决不同问题方面变得非常有前景，比如生物医学设备缺乏可追溯性或设计更灵敏的生物传感器。其中，我们发现了类弹性蛋白重组体（ELR），它可以被设计成自组装成各种结构，如水凝胶。此外，它们还可以与其他蛋白质聚合物，如丝绸，结合形成类丝弹性蛋白重组体（SELR），兼具两种蛋白质的特性。在这项工作中，由于其重组性质，我们将两种不同的荧光蛋白（FP），即绿色的海肾增强型绿色荧光蛋白和近红外的eqFP650，融合到一种能够通过物理交联形成不可逆水凝胶的SELR上。这些重组体发出的荧光与单个FP相似，并且通过改变SELR-FP的浓度，它们能够形成具有不同硬度（G' = 60 - 4000 Pa）的水凝胶。此外，SELR-eqFP650的吸收光谱显示出一个与SELR-海肾增强型绿色荧光蛋白的发射光谱有很大重叠的峰。因此，由于在任何温度下丝域的堆积以及在转变温度以上类弹性蛋白块的聚集，当两个分别含有不同FP的SELR分子相互作用时，这使得能够发生Förster共振能量转移（FRET）。通过不同方法研究了这种效应，根据用于计算的技术，观察到FRET效率为0.06 - 0.2。因此，SELR与FP的结合产生了创新的生物学应用，例如提高用于组织工程的基于SELR的水凝胶的可追溯性、生物传感器的开发以及新型FRET对的FRET效率预测。
