Rapid RNase inhibitor production to enable low-cost, on-demand cell-free protein synthesis biosensor use in human body fluids.

Autor: Soltani M; Department of Chemical Engineering, Brigham Young University, Provo, Utah, USA., Hunt JP; Department of Chemical Engineering, Brigham Young University, Provo, Utah, USA., Bundy BC; Department of Chemical Engineering, Brigham Young University, Provo, Utah, USA.
Jazyk: angličtina
Zdroj: Biotechnology and bioengineering [Biotechnol Bioeng] 2021 Oct; Vol. 118 (10), pp. 3973-3983. Date of Electronic Publication: 2021 Jul 23.
DOI: 10.1002/bit.27874
Abstrakt: Human body fluids contain biomarkers which are used extensively for prognostication, diagnosis, monitoring, and evaluation of different treatments for a variety of diseases and disorders. The application of biosensors based on cell-free protein synthesis (CFPS) offers numerous advantages including on-demand and at-home use for fast, accurate detection of a variety of biomarkers in human fluids at an affordable price. However, current CFPS-based biosensors use commercial RNase inhibitors to inhibit different RNases present in human fluids and this reagent is approximately 90% of the expense of these biosensors. Here the flexible nature of Escherichia coli-lysate-based CFPS was used for the first time to produce murine RNase Inhibitor (m-RI) and to optimize its soluble and active production by tuning reaction temperature, reaction time, reduced potential, and addition of GroEL/ES folding chaperons. Furthermore, RNase inhibition activity of m-RI with the highest activity and stability was determined against increasing amounts of three human fluids of serum, saliva, and urine (0%-100% v/v) in lyophilized CFPS reactions. To further demonstrate the utility of the CFPS-produced m-RI, a lyophilized saliva-based glutamine biosensor was demonstrated to effectively work with saliva samples. Overall, the use of CFPS-produced m-RI reduces the total reagent costs of CFPS-based biosensors used in human body fluids approximately 90%.
(© 2021 Wiley Periodicals LLC.)
Databáze: MEDLINE
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