Application To Functional Proteomics

In order to evaluate the performance of the cell-free method in high-throughput screening, we carried out parallel protein syntheses from 27 genes originating with E. coli cells carrying the cDNAs [7]. In 50 out of the total 54 cases (authentic and fusion proteins with GST), a clearly visible CBB-stained protein band was obtained. The yield after 36 h of incubation was estimated by densitometric scanning of the bands using BSA as the standard. It was from 0.1 to 2.3 mg/mL of the reaction volume in the dialysis cup mode reaction (CFCF) (Table 44.1). Some gene products were recovered in soluble forms in the supernatant (S) and some others in the precipitate phase (P) after centrifugation at 30,000 g for 15 min. It is worth mentioning here that we could not detect any dependency of the productivity and solubility of the proteins on the gene sources. Furthermore, the system had little preference in codon usage, which is a prerequisite for genome-wide protein expression. In fact, we could express the quality of malaria proteins from 76% AT-rich cDNAs of P. falciparum [10]. The most important requirement for an expression system, however, is that it produces a quality product. To verify the quality of proteins and the general applicability of the wheat germ cell-free system, we tested a subset of product proteins for their functional activity (Figure 44.2). PHOT1 gene from A. thaliana codes for a protein of 120 kDa which noncovalently binds to flavin mononucleotide (FMN) and presumably acts as a chro-mophore in light-dependent autophosphorylation [11]. As shown in Figure 44.2A, the protein that was responsible for the blue-light-responsive activity could be produced only when the translation was carried out in the presence of FMN [12]. The result

TABLE 44.1. An Example of Proteins Synthesized in the PCR-Directed Wheat Germ

Cell-Free System

TABLE 44.1. An Example of Proteins Synthesized in the PCR-Directed Wheat Germ

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