PURE system is a cell-free protein synthesis system reconstituted from only factors involved in protein synthesis in E. coli. PURE system can be used for synthesis of proteins with disulfide bridges in active form by adding DsbC from E.coli as protein disulfide isomerase (PDI). Here, we will present the results confirming that human protein disulfide isomerase (hPDI) and related proteins also can be used in PUREfrex® (improved version of PURE system) for application to wide range of protein synthesis with complex structures or from higher organism having disulfide bonds. As a model protein, we synthesized tissue plasminogen activator (tPA) with 9 disulfide bonds. The tPA synthesized by adding hPDI and GSSG to PUREfrex® using GSH as a reducing agent showed dose-dependent activity along with the amount of added hPDI. On the other hand, without GSSG, the tPA synthesized by adding hPDI and hEro1a (PDI oxidase) showed same level of high activity. Thus, it was found that either GSSG or hEro1a was necessary for hPDI to work. In comparison of hPDI and DsbC under the existence of GSSG, hPDI showed almost same effect with DsbC. As a result, it was confirmed that the proteins with disulfide bridges were effectively synthesized in active form even with hPDI adjusting redox conditions or adding hPDI oxidase. We will also report other examples here.