Translational Detection of Nonproteinogenic Amino Acids Using an Engineered Complementary Cell-Free Protein Synthesis Assay

Abstract

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.

Translational Detection of Indole by Complementary Cell-free Protein Synthesis Assay

“…In addition to being the building blocks of proteins, the synthesis and degradation pathways of amino acids are closely interlinked with the metabolism of diverse compounds. Therefore, these compounds can be enzymatically converted into proteinogenic amino acids for their measurement by the CCFPS assay (Lim et al, 2020).…”

A Dual-Channel Sensor Array: Ultraviolet Absorption and Surface Potential Measurements for Discrimination of Amino Acids Based on Gold Nanorods

“…Amino acids are fundamental and important biomolecules, which are responsible for many tasks of cells, such as protein synthesis, signal transmission, and so on. − The occurrence of certain diseases is closely related to the abnormality of amino acid concentration, such as elevated amino acid concentration in blood or urine as a clinical indicator of metabolic diseases. − Therefore, the detection and identification of amino acids are of far-reaching significance in clinical diagnosis and biochemical research.…”

“…At present, amino acid detection methods include targeted and nontargeted metabolomics. The specific detection methods include chromatography, capillary electrophoresis, and other methods. − Although these methods can detect single amino acids with high sensitivity and selectivity, most of these methods require complex equipment, long processing time, and so on . It is worth mentioning that these methods all need specific recognition receptors for one target, i.e., the “lock-key” mode, which limits the simultaneous discrimination of multiple targets.…”

“…7−13 Although these methods can detect single amino acids with high sensitivity and selectivity, most of these methods require complex equipment, long processing time, and so on. 3 It is worth mentioning that these methods all need specific recognition receptors for one target, i.e., the "lock-key" mode, which limits the simultaneous discrimination of multiple targets. In order to tackle these issues, inspired by the mammalian olfactory/gustatory systems, sensor arrays, which are called "chemical nose/tongue", have become a powerful means for detecting and distinguishing multiple analytes.…”