Recombinant expression systems

In particular, the availability of such bacterial biocatalysts in the form of recombinant expression systems [136] in combination with simplified purification protocols opened up this methodology for large-scale applications [204]. 

As a result, protein molecules can be constructed in quantities well beyond those accessible in cell-free recombinant expression systems, which incorporate residues or linkages that cannot be specified by the genetic code. Very large chimeric fusion proteins can be made, and since the reaction conditions are generally quite mild, this can be done under circumstances where the individual protein modules retain their native conformation at all... 

The detailed characterization of hydroxynitrile lyases from Sorghum hicolor (E.C. 4.1.2.11) and Linum usitassimum (E.C. 4.1.2.37) has been hampered for a long time due to the lack of a recombinant expression system. Therefore our studies were focused on cloning of the coding genes, recombinant expression, and characterization of these enzymes. 

The N-terminal region of fibrillin-1 (the first 29 residues after signal peptide) contains largely basic residues (estimated pi, 11.1). There is an N-terminal putative furin cleavage site at the N-terminus (RAKRjR residues 41-45). Recombinant secreted N-terminal fibrillin-1 can be furin processed (Raghunath et al., 1999 Ritty et al., 1999 Wallis et al., 2003). In our mammalian recombinant expression system, a significant level of... 

The production of antibodies in transgenic plants was introduced in 1989 by Hiatt, Cafferkey, and Bowdish.45 One of the important justifications of the choice of this host organism is the low cost of plant maintenance. Another advantage over other recombinant expression systems is the ability to assemble full-length heavy chains with light chains to form full-length antibody efficiently.46,47... 

One cannot over-emphasize the importance of the enzyme preparation in the ability to develop an assay. First, purity is important because even the slightest contamination with another enzyme can lead one to screen with a measurement of the wrong activity. Specific reference inhibitors can be used to establish that the observed catalytic activity is the correct one. Of course, when working with novel targets, such reference inhibitors may not exist. With recombinant expression systems, one can generate catalytically inactive mutations to establish that the host cell is not the source of a contaminating phosphatase or kinase. In the end, however, the key requirement is to have an extremely pure and highly active enzyme preparation. 

Although the use of recombinant genes for protein production is often very successful, problems can arise. Some genes or cDNAs are not expressed well in the commonly used vector/host systems. Occasionally, the overproduced proteins do not fold properly and aggregate into insoluble inclusion bodies in which the recombinant protein is denatured. Posttranslational modifications, such as phosphorylation or glycosylation, that occur in the native cells and are important for the function of the protein may not occur in the recombinant expression system. 

Kenakin, T.P., 1996. The classification of seven transmembrane receptors in recombinant expression systems. Pharmacol. Rev. 48, 413-463. 

Table 5.3 Qualitative ranking to illustrate strengths and weakness of recombinant expression systems...

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