Cloned gene expression translation

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. 

Once a gene is cloned it is necessary to convert the information contained in it into a functional protein. There are a number of steps in gene expression (i) transcription of DNA into mRNA (ii) translation of the mRNA into a protein sequence and (iii) in some instances, post-translational modification of the protein. In discussing these steps in more detail, expression of a cloned insulin gene will be used as an example. 

Figure 2 Comparison of cloning and expression methods. In the conventional strategy (left), dehydrogenase genes obtained by PCR amplification of the original source DNAs are cloned into overexpression plasmids and verified by sequencing. Those with the desired structure are individually transformed into suitable host strains and the proteins are obtained, either as crude extracts or as purified samples. In the proposed streamlined approach (right), full-length dehydrogenase genes obtained by chemical synthesis are used directly in coupled transcription/translation reactions to obtain the proteins of interest.

Genes can similarly be cloned and expressed in eukaryotic cells, with various species of yeast as the usual hosts. A eukaryotic host can sometimes promote post-translational modifications (changes in protein structure made after synthesis on the ribosomes) that might be required for the function of a cloned eukaryotic protein. 

Gene expression. Synthesis of a functional protein depends on transcriptimi of the gene, translation of the mRNA, often processing of the mRNA, and often post-translational processing of the initial translation product (see Post-translational modification of proteins). TVanscription of a cloned insert requires the presence of a promoter which is recognized by the host RNA polymerase. Translation requires a ribo-... 

The selection of an expression system for a cloned gene depends on many factors host/vector stability, expression level, ease of scale-up, post-translational modifications (glycosylation, secretion, amidation, folding, proteolysis, phosphorylation, etc.), product immunogenecity, and heterogenicity. 

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