Expression vectors stabilization

As it is imperative that the plant-derived hiopharmaceutical product must be obtained repeatedly and on a consistent basis, a master cell culture bank, seed bank for transgenic plants, or virus seed stock for transient expression systems must be constantly maintained. Storage conditions must therefore he optimized to prevent contamination and ensure viability. Both transgene stability (e.g., reversion to wild type or sequence drift of plant virus expression vectors) and protein expression levels must be monitored in a representative plant of a given bank or stock to minimize any possible variation in expression levels that may affect safety and consistency of the hnal product. A program that monitors lot-to-lot consistency of the hiochemical and biological properties by comparing the product with appropriate in-house reference standards could he implemented as a fundamental component of product development. 

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

In most cases, the DNA inserted into the MCS for expression is not a genomic gene with the original exon/intron configuration, but a cDNA. In exceptional cases, antisense RNA or ribozymes may be expressed. cDNA lacks intronic sequences, but still has the 5 and 3 UTR sequences. As discussed above, the presence of the 3 UTR may reduce the stability of mRNA transcribed from the cDNA insert. Furthermore, the length and secondary structure of the 5 UTR may influence the efficiency of translation. Therefore, it is generally recommended to use cDNA with only short 5 and 3 UTR sequences for expression with expression vectors. 

Fig. 12.1 Plasmid stability of the IL-4 v expression vectors pR021.1.0 (T7-promoter based) and pR02.1.0 (T5 promoter-based). For details, see Table 12.2.

Endo and co-workers at Ehime University, Matsuyama, Japan, have led the development of the most promising eukaryotic cell-free system to date, based on wheat embryos. A significant advance made by this group was the development of pEU expression vectors that have overcome many of the difficulties associated with mRNA synthesis for translation in a eukaryotic system [8]. In addition to extensive optimization of reaction conditions that have seen improvements in protein synthesis rates, Endo and colleagues have improved wheat extract embryo preparation protocols to enhance the stability of these systems to a remarkable extent [9]. When coupled with the dialysis mode of reaction, Endo et al. were able to maintain translational activity in a coupled transcription/ translation wheat embryo reaction for 150 hours, producing 5 mg of enzymatically active protein per mb reaction mixture [10]. This again represents a serious alternative to in vivo methods of large-scale protein production. 

Most proteins used for therapeutic purposes In humans or animals are secreted glycoproteins stabilized by disulfide bonds. When researchers first tried to synthesize such proteins using plasmid expression vectors in bacterial cells, the results were disappointing. In most cases, the proteins were not secreted (even when a bacterial signal sequence replaced the normal one) instead, they accumulated in the cytosol and often in a denatured state, in part owing to the lack of disulfide bonds. After It became clear that disulfide-bond formation occurs spontaneously only in the ER lumen, biotechnologists eventually developed expression vectors that can be used In animal cells (Chapter... 

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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