The activity of sweet taste receptor (heterodimeric T1R2 and T1R3) can be modulated by sweet regulators. The compound amiloride can inhibit the sweet sensitivity of the human sweet taste receptor. This study describes the species-dependent regulation of the response of sweet taste receptors by this sweet inhibitor. Amiloride inhibited the sweet taste response of humans and mice but not that of squirrel monkeys. Using human/squirrel monkey/mouse chimeric T1R2 and T1R3 receptors as well as the agonist perillartine (which can activate the single heptahelical domain of T1R2), we found that the heptahelical domain of T1R2 is the molecular determinant that mediates the species-dependent sensitivity to this sweet regulator. Compared to the sweet inhibitor lactisole (which acts on T1R3), amiloride has a different allosteric binding site on the sweet receptor, which is important new information for the design of novel sweet taste modulators that act on T1R2.

ABSTRACT. The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS) 1 , L2: (GGGGS) 2 , and L3:(GGGGS) 3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins 19361Linker length affects HSA-fused ONC in P. pastoris ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 19360-19370 (2015) were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.

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