Recombinant gene expression system

Gene Expression Systems. One of the potentials of genetic engineering of microbes is production of large amounts of recombinant proteias (12,13). This is not a trivial task. Each proteia is unique and the stabiUty of the proteia varies depending on the host. Thus it is not feasible to have a single omnipotent microbial host for the production of all recombinant proteias. Rather, several microbial hosts have to be studied. Expression vectors have to be tailored to the microbe of choice. 

An additional screening test for TCDD-like (aryl hydrocarbon receptor, AhR, active) chemicals has been developed (Garrison et al. 1996) and is available commercially (Anonymous 1997). Dubbed the CALUX (for chemically activated luciferase gene expression) system, the assay is based on recombinant cell lines into which researchers have inserted a firefly luciferase gene. When exposed to dioxin-like compounds, the recombinant cells luminesce. The method is sensitive to ppt levels of 2,3,7,8-TCDD equivalents in blood, serum, and milk (Anonymous 1997). Samples testing positive can be subjected to more definitive and specific analytical testing. 

Over several decades, multiple vector systems for recombinant gene expression in E. coli have been developed. Modem vectors suitable for recombinant protein production vary in the used promoter system in the presence or absence of coding sequences for affinity tags upstream or downstream of the multiple cloning site (MCS) and of sequences coding for leader peptides for the protein export. Moreover, different origins of replication (ori), antibiotic selection marker genes and MCS are used. 

Only a few years ago, first studies were started using the xylose-inducible plasmid-borne gene expression system for the production and secretion of recombinant proteins by B. megaterium. The production and secretion of the high molecular mass dextransucrase DsrS (Mr = 188,000) from Leuconostoc mesenteroides... 

Fuentes, A. Ramos, P.L. Ayra, C. Rodriguez, M. Ramirez, N. Pujol, M. Development of a highly efficient system for assessing recombinant gene expression in plant cell suspensions via Agrobacterium tumefaciens transformation. Biotechnol. Appl. Biochem. 2004, 39, 355-361. 

Several E. coli heterologous gene expression systems based on the tac, T7, and Hlac promoters have been used successfrilly to obtain recombinant archaeal histones, by following the commercial protocols specified for each expression system. The purification protocol detailed above for native archaeal histones also works well for recombinant archaeal histones, although the protease inhil >itor phenylmethylsulfonyl fluoride (PMSF) must be added to a final concentration of 0.1 mAf during the DNase I digestion. Almost 100% purity can be achieved from E. coli crude extracts. 

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. 

Volume 306. Expression of Recombinant Genes in Eukaryotic Systems Edited by Joseph C. Glorioso and Martin C. Schmidt... 

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