(Invited) A Protein-Based Artificial Receptor for Insulin Sensing
Authors: Funabashi Hisakage , Shigeto Hajime 
Published: December 2020

Insulin is one of the most important hormones that controls the glucose level in blood. Therefore, the investigation of insulin is very important for the diabetic research. There have been many reports analyzing the role of insulin with insulin detecting methods. Most of these methods utilized antibodies or aptamers for insulin recognition. Although these sensors are highly sensitive and specific to insulin attributed to the target recognition ability of antibodies or aptamers, there is a possibility to detect physiologically inactive insulin such as partially digested or mutated insulin because the antibodies or the aptamers recognize only a part of insulin molecule. However, what we need is to measure the concentration of functionally active insulin for accurate investigation in our body. Therefore, it is necessary to develop a novel method based on a new target recognition mechanism for the accurate evaluation of insulin.
To meet the demand, we have paid attention to the mechanism of recognition in an insulin receptor, because the recognition by the insulin receptor is the first step and one of the most essential processes for insulin to exhibit its function in nature. The insulin receptor is a dimer protein and each monomer contains the carboxy-terminal α-chain (αCT) segment and the ﬁrst leucine-rich repeat with the cysteine-rich (L1CR) domain as an insulin recognition site. The insulin molecule is captured by the αCT segment from one monomer and the L1CR domain from the other monomer. However, a part of insulin receptor is buried in a cellular membrane and thus it is difficult to use the receptor in a soluble format. Hence, we have developed a protein-based artificial receptor which is soluble in aqueous environment. In this presentation, we demonstrate the applications of the artificial receptor for biosensing such as online monitoring of insulin secretion from drug-stimulated living cells and for imaging analysis of insulin secretion in a single cellular level.
The genes of the αCT segment and the L1CR domain in the primary binding site on the insulin receptor were connected with an appropriate length of peptide linker to be the main component of protein-based artificial insulin receptor. To detect the structural change of the artificial receptor in response to the insulin recognition, the genes of a bioluminescent protein (Nanoluc) and a ﬂuorescent protein (YPet) were further connected. By connecting these proteins, the structural change could be evaluated by a bioluminescence resonance energy transfer (BRET) system. The genetically fused artificial protein termed as NαLY, was expected to work as a biosensing protein probe, exhibiting BRET signal in response to insulin. The gene was successfully expressed utilizing mammalian cells and the expressed protein was purified utilizing His-tag affinity in aqueous solution.
The feasibility of insulin detection with NαLY was confirmed by measuring luminescence spectra. The luminescence spectra indicated a clear BRET signal in response to insulin. The emission being attributed to Nluc decreased and the emission from YPet increased. The BRET units were proportional to the concentration of insulin, and thus we concluded that the artificial receptor-based insulin sensing probe has an insulin recognition ability presumably reflecting that of the insulin receptor and works as a biosensing probe for insulin. Then, the artificial-receptor-based biosensing probe was applied for the online monitoring of insulin secretion. When the probe was added in the culture medium, it was proved that a time-course in BRET signal reflected the insulin secreting response of drug-stimulated pancreatic-β-cells (MIN6 cell line). To analyze the secretion of insulin from a single-living cell, insulin sensor cells which express the artificial-receptor-based biosensing probe on a cellular surface were fabricated. The sensor cells were then co-cultured with insulin-secreting cells. Imaging analysis revealed that some of the sensor cells nearby pancreatic β cells showed a spike-shaped BRET signal response when co-cultured MIN6 cells were stimulated by glucose.
From these results, it is concluded that the protein-based artificial receptor has a great potential to be a platform to create a novel biosensing probe for insulin sensing.