Translation clone selection

Cell-free translation system, used for the identification of cloned genes and gene expression, has been investigated extensively as a preparative production system of commercially interesting proteins after the development of continuous-flow cell-free translation system. Many efforts have been devoted to improve the productivity of cell-free system [1], but the relatively low productivity of cell-free translation system still limits its potential as an alternative to the protein production using recombinant cells. One approach to enhance the translational efficiency is to use a condensed cell-free translation extract. However, simple addition of a condensed extract to a continuous-flow cell-free system equipped with an ultrafiltration membrane can cause fouling. Therefore, it needs to be developed a selective condensation of cell-free extract for the improvement of translational efficiency without fouling problem. 

The first step in biopharmaceutical development is the selection of a clone in a specific cell line. Whole-mass analysis, if possible, is a fairly simple and powerful tool at this stage to verify the successful expression and translation of the desired protein. VanAdrichem et al.65 described the use of MALDI MS to monitor protein expression in several mammalian cell lines like CHO DXB11, CHO SSF3, and hybridomas. Quantitative MALDI-TOF MS measurements of an IgG antibody and insulin during large-scale production in hybridoma cells were comparable to affinity chromatography results. 

PAL cDNA clones have been isolated from a number of species (Table 1). The first PAL cDNA clone was isolated from French bean suspension culture cells treated with fungal elicitor (Edwards et al., 1985). The clones were preselected by differential hybridisation to RNA from non-treated and elicitor-treated suspension culture cells. Further identification involved in vitro translation of hybrid-selected RNA and immunoprecipi-tation (Edwards et al., 1985), using an antibody raised against the purified bean PAL enzyme (Bolwell etal., 1985). This work identified pPAL5 as a PAL cDNA clone. 

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