Introduction

In vitro expression technologies offer significant time savings over cellular approaches and continue to rapidly expand. Moreover, cell-free protein synthesis has become a powerful alternative to cell-based methods in producing proteins on a preparative scale (1,2). Continuous-exchange cell-free (CECF) protein synthesis has been used for the preparative-scale production of proteins from a circular plasmid. In this method, incubation of the translation mixture in a simple dialysis bag was proposed for replenishing substrates and removing low molecular weight byproducts by diffusion exchange across the membrane during protein synthesis. The CECF system based on Escherichia coli extracts has recently been launched into the market by Roche Diagnostics GmbH (RTS 500, RTS 9000, RTS ProteoMaster; Mannheim, Germany). Although this RTS ProteoMaster instrument is favorable for obtaining a high yield of protein through controlled shaking and temperature control, the instrument is relatively expensive.

PCR-generated DNA (i.e., linear template) has been routinely used in the cell-free protein synthesis system in a high-throughput format as template DNA because of its simple preparation. However, it still has not been used for preparative-scale protein production due to the significantly lower expression level from it compared with a circular plasmid.

In this article, for the purpose of promoting rapid soluble protein production with a high yield from PCR-generated DNA, a simple CECF system was constructed using an RNase E-deficient and molecular chaperone-enriched extract.

Materials and Methods

Preparation of S30 Extracts from BL21 (DE3) and Its Derivative Strains

A normal S30 extract and a molecular chaperone-enriched S30 extract were prepared from E. coli BL21 (DE3) and its derivative strains, which have genes of molecular chaperone or disulfide isomerase, as described previously (3). A mixture of the molecular chaperone-enriched S30 extracts was prepared by simple mixing of these extracts. The extracts used for the preparation of the mixture of the molecular chaperone-enriched S30 extract are presented in (Table 1). The volumetric mixing ratio of the mixture is as follows:

S30BL:S30BL/Dna:S30BL/GroE:S30BL/DsbC = 67:82:17:1

The volumetric mixing ratio for the preparation of the mixture of a molecular chaperone-enriched extract was determined experimentally to maximize the activity of erythropoietin (EPO) translated as described previously (3).

Preparation of S30 Extracts from BL21 Star (DE3) and Its Derivative Strains

S30 extracts derived from E. coli BL21 Star (DE3) [F- ompT hsdS_B (r_B-m_B-) gal dcm rne131 (DE3)] (Invitrogen, Carlsbad, CA, USA) and its derivative strains, which also have genes for molecular chaperone or disulfide isomerase, were prepared for use as an RNase E-deficient S30 extract and an RNase E-deficient and molecular chaperone-enriched S30 extracts as described previously (3). It was reported that the use of extract derived from BL21 Star (DE3) was favorable in obtaining a high yield of protein from a linear template in the batch-type cell-free system (4). A mixture of RNase E-deficient and molecular chaperone-enriched S30 extracts was prepared by simple mixing of the RNase E-deficient S30 extract and RNase E-deficient and molecular chaperone-enriched S30 extracts. The extracts used for the preparation of the mixture of RNase E-deficient and molecular chaperone-enriched S30 extracts are presented in (Table 1). The same volumetric mixing ratio for the preparation of the mixture of an RNase E-deficient and molecular chaperone-enriched extract was applied. The volumetric mixing ratio of the mixture is as follows:

S30Star:S30Star/Dna:S30Star/GroE:S30Star/DsbC = 67:82:17:1

Preparation of Linear Templates for Cell-free Protein Synthesis

To generate the linear templates, two successive PCRs were performed. In the first round of gene-specific PCR, defined overlapping regions with the sequence in the DNA fragments required in the subsequent overlap extension PCR were added to the first-round PCR products using gene-specific primers. In a subsequent overlap extension PCR, the regulatory elements necessary for expression in a prokaryotic system based on T7 polymerase (e.g., the T7 promoter), the ribosomal binding site, and the T7 terminator were introduced into the second-round PCR products. Chloramphenicol acetyl transferase (CAT) and EPO were chosen as model proteins. Gene-specific primers containing both an overlapping region with the sequence in the DNA fragment and gene-specific priming sequence were designed for the first gene-specific PCR ((Table 2)). To exclude the possibility of expression from the template plasmid of the first gene-specific PCR, the cloning plasmid without any regulatory elements for protein expression or the plasmid derived from a eukaryotic expression vector, which cannot support the expression in the prokaryotic expression system, was used in the first gene-specific PCR. The CAT gene in pK7-CAT (1) was subcloned to pUC 19 using NdeI/SalI sites. The plasmid generated was named pUC19-CAT and was used as a template plasmid for the first CAT gene-specific PCR. p64T-EPO (5) was used as a template plasmid for the first EPO gene-specific PCR.