The expression of properly folded proteins has long been one of the central issues in modern biotechnology. Especially, the importance of developing an efficient and robust protein expression method has become greater than ever, due to the vast amounts of DNA sequence information that have been accumulated by the rapidly progressing genome sequencing projects. The excellent speed and flexibility of cell-free protein synthesis have led many researchers to seek to use this method to produce and engineer protein molecules as an alternative or complementary technology to the in vivo expression of proteins. However, the proper folding of the cell-free expressed protein still remains one of the major hurdles to overcome before the practical use of this technique can be seriously envisaged. In this context, the expression of eukaryotic proteins with multiple disulfide bonds represents a particular challenge, due to the complications involved in optimizing the reaction conditions for the proper formation of disulfide bonds as well as the efficient translation of protein molecules. While the formation of disulfide bonds requires an oxidative environment, conventional reactions of cell-free protein synthesis are conducted under reducing conditions. The reaction mixture used for cell-free protein synthesis typically contains 2 – 3 mM of dithiothreitol (DTT), and thus naturally, disulfide bonds are not formed between the cysteine residues of the expressed proteins. Moreover, the formation of disulfide bonds is not brought about by simply adding oxidizing agents (such as oxidized glutathione), because of the presence of multiple disulfide-reducing enzymes in the cell-free extract (generally referred to as the E.coli S30 extract). In fact, the added oxidizing agents do not contribute to the maintenance of a high redox potential, as they are rapidly reduced by these reducing enzymes. Also Derman demonstrated that mutant trxB, one of the major reductase in E.coli cytoplasm, strain can produce active urokinase in their cytoplasm.

In a recently published report, however, Kim and Swartz demonstrated that the activities of the endogenous reducing enzymes could be effectively repressed by treating the entire S30 extract with iodoacetamide (IAM), a potent sulfhydryl-blocking agent. In their study, the protease domain of murine urokinase that was expressed in the IAM-treated S30 extract exhibited enzymatic activity, indicating that the disulfide bonds were correctly formed within the structure of the expressed protein. In our experiments designed to express the recombinant tissue plasminogen activator in the S30 extract derived from the E.coli strain, BL21(DE3), however, in turned out that the translational activity of the S30 extract was significantly impaired upon the addition of IAM. The amount of synthesized rPA in the IAM-treated S30 extract was less than 50% of that of the control extract without any chemical treatment. The reason is presumed that essential cysteine residue in Creatine kinase is inactivated by treatment of IAM.

In this study, we describe an alternative method of repressing the reducing activity of the S30 extract without sacrificing its translational activity. Instead of the covalent blocking of reducing enzymes, the S30 extract was incubated with 10 mM of oxidized glutathione prior to its use for cell-free protein synthesis. Such a pre-treatment led to the exhaustion of the reducing power of the extract without deteriorating its translational activity. When the resulting S30 extract was used for the cell-free expression of rPA in the presence of glutathione buffer, the redox potential of the reaction mixture remained stable, and the expressed protein exhibited the enzymatic activity of rPA. It was also found that the generation of rPA was especially effective when the cell-free protein synthesis reaction was conducted in batch configuration, which implies that the developed method can be applied to the parallel expression of multiple proteins containing disulfide bonds.

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