Development Of Wheat Germ Cellfree Protein Synthesis System

We have been actively pursuing ways to improve the wheat germ cell-free translation system through its understanding from both fundamental and technological viewpoints. Recently, we found that the conventional wheat germ extracts contain the RNA N-glycosidase tritin and other inhibitors of translation such as thionin, ribonu-cleases, deoxyribonucleases, and proteases which are suspected of being involved in a suicide system targeting at translational machinery. We also ascertained that those inhibitors originate from the endosperm [5, 6]. Extensive washing of wheat embryos could eliminate those endosperm contaminants to produce clean embryos for extracts with a high degree of stability and activity [6]. In fact, this new translation system proved stable not only in its activity and longevity but also in storage; it can be stored for years without loss of activity, even in the lyophilized state. In order to maximize the throughput of the system for practical use, other elemental techniques were developed. They are (i) eliminating both the 5'-mGpppG (cap) and poly(A)-tail (pA) by optimizing the 5'- and 3'-UTRs of mRNA, thereby increasing translation initiation and the stability of the template [7]; (ii) designing the split-primer for PCR to generate transcription templates that can minimize artificial generation of the products [7], a technique that permits high-throughput construction of DNA templates directly from E. coli cells carrying cDNAs, with the time-consuming cloning steps bypassed; (iii) constructing an expression vector pEU [7] specialized for the mass production of proteins, which reduces introduction of inevitable mutation during the PCR and abates the cost; (iv) developing the transcription and translation reaction in one tube in which no mRNA purification step is needed [8]; and (v) inventing the bilayer reaction that enables us to perform CFCF mode translation without using a membrane for the high-throughput production [9]. Combining all these elemental techniques, we could establish two cell-free protocols for practical use (Figure 44.1). One of the two is the protocol for materializing genetic information in parallel, which consists of (i) in silico selection of suitable genes from the database, (ii) construction of templates for transcription by the split-PCR, (iii) transcription, and (iv) the bilayer translation in which the solution resulting from transcription is directly used as mRNA source. For the production of proteins with such tags as for purification or for the reporter or for both, DNA constructs can be generated by the split-PCR as well. The other protocol is for massive preparation (bold arrow), which consists of (i) selection of suitable gene products from the parallel production described above and subsequent functional

FIGURE 44.1. Procedures for protein synthesis using the wheat germ cell-free system.

screening, (ii) cloning of the genes into pEU and transcription of mRNA, (iii) fine tuning of translational conditions such as the concentrations of pertinent ions, and (iv) protein production that incorporates either the bilayer or dialysis methods. Proteins with a desired purification tag can easily be affinity purified, and the tag portion can be removed by proteolytic cut at a designed linker sequence if desired.

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