PUREfrex® is a cell-free protein synthesis system reconstituted from only factors involved in protein synthesis in E. coli. Following the condition in cytosol, DTT is applied as a reducing agent in PUREfrex®. However, it was turned out that the reaction mixture was oxidized after long incubation. For example, when PUREfrex® reaction mixture was incubated without template DNA at 37oC for 24 hours, EF-Tu and some other translation factors showed different mobility in SDS-PAGE under non-reduced conditions. The reaction mixture after long incubation was applied to MS analysis, and it detected the peptide derived from translation factors with free Cys attached to its internal Cys residues. Also, quantitative analysis of SH residues in reaction mixture with Elman’s reagent suggested that SH residues were remarkably decreased under the detection limit after 24 hours incubation. Those phenomena was not observed when GSH was used as a reducing agent. When the reducing agents were changed, the amount of protein synthesized barely changed, but there were influence on disulfide bridge formation. As a conclusion, it suggests that appropriate reducing agent should be used for each protein in preparation with PUREfrex®.