Recombinant expression vectors

Once the recombinant expression vectors containing the cDNA inserts are produced, they are used to transform bacteria (or other host cells) and produce cDNA (expression) libraries. Expression libraries may be used to produce recombinant proteins that in some cases have significant advantages over isolating them from natural sources ... 

B. Lawlis, W. Rastetter, and R. Snedecor, Recombinant process for preparing Lramino adds, recombinant expression vectors and transformed microorganisms for use in the process, European Patent Application No. 84305532.8, 1985. 

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. 

A vector for in vitro expression of DNA inserts as RNA transcripts can be constructed by putting a highly efficient promoter adjacent to a versatile cloning site. Figure 13.15 depicts such an expression vector. Linearized recombinant vector DNA is transcribed in vitro using SPG RNA polymerase. Large amounts of RNA product can be obtained in this manner if radioactive ribonucleotides are used as substrates, labeled RNA molecules useful as probes are made. 

Recombinant plant viruses have been used as expression vectors because the infections are rapid and systemic, leading to high levels of recombinant protein produc-... 

If a recombinant protein is developed and expressed for research purposes, the most labor- and cost-intensive part of the project normally lies with the upstream tasks, i.e. cloning, expression vector design, sequencing, transformation and selection. Once a suitable expresser strain or plant line is available, it is often sufficient to visualize expression of the recombinant protein, verify its activity, study its biological... 

The desired gene/cDNA is normally amplified, sequenced and then introduced into an expression vector that facilitates its introduction and expression (transcription and translation) in an appropriate producer cell type. All recombinant therapeutic proteins approved to date are produced in E. coli, S. cerevisiae or in animal cell lines (mainly CHO or BHK cells). The general... 

Yeast expression vectors have been among those most commonly used since the beginning of gene technology. Vectors based on baker s yeast, Saccharomyces cerevisiae, have been especially popular for robust expression of many types of recombinant proteins [90]. For instance, the first commercially available recombinant vaccine, the hepatitis B surface antigen vaccine, was produced from an S. cerevisiae vector [91]. Many other recombinant proteins have also been efficiently expressed in yeast including al-Antitrypsin [92], insulin [93], Epstein-Barr virus envelope protein [94], superoxide dismutase [95] and interferon-a [90]. 

Vectors for recombinant DNA and cloning Plasmid pBR322 Restriction site Replication origin Resistance to antibiotic(s) Expression vector also requires Promoter Shine-Dalgarno sequence Other vectors phage, YACs... 

Insect cells in culture are also hosts for recombinant protein production. Production of recombinant proteins in the baculovirus expression vector system is the most common system. Titers of recombinant protein as high as 11 g/L have been obtained. 

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