Foreign Protein Stability

Extensive research has been carried out into the molecular aspects of foreign protein production in whole plants to enhance the yield, quality and stability of the product and to facilitate protein separation and purification from the biomass [3, 6, 9], In contrast, comparatively little research has been undertaken to investigate the... 

As indicated in Table 2.1, most of the promoters used in plant tissue culture have been based on the constitutive cauliflower mosaic virus (CaMV) 35S promoter. In contrast, inducible promoters have the advantage of allowing foreign proteins to be expressed at a time that is most conducive to protein accumulation and stability. Although a considerable number of inducible promoters has been developed and used in plant culture applications, e.g. [32-37], the only one to be applied thus far for the production of biopharmaceutical proteins is the rice a-amylase promoter. This promoter controls the production of an a-amylase isozyme that is one of the most abundant proteins secreted from cultured rice cells after sucrose starvation. The rice a-amylase promoter has been used for expression of hGM-CSF [10], aranti-trypsin [12, 29, 38, 39] and human lysozyme [30]. 

The presence of foreign protein in the medium of plant cultures does not necessarily mean that all or even most of the product can be recovered from the medium. In many expression systems where an appropriate signal sequence has been used, considerable amounts of foreign protein remain within the plant cells and/or tissues. For example, in a comparison of IgG antibody production in tobacco cell suspension and hairy root cultures, a maximum of 72% of the total antibody was found in the medium of the suspension cultures whereas only 26% was found in the medium of the hairy root cultures [17]. This result could indicate that secretion and/or transport across the cell wall was slower in the hairy roots alternatively, it could indicate poorer stability of the secreted protein in the hairy root medium. If foreign proteins are to be purified from the medium, improved secretion and extracellular product stability are desirable. 

There have been many reports from several groups that plant culture medium is not conducive to protein stability, and that the retention of secreted proteins in culture media can be very poor [10, 11, 17, 40, 60, 63-66]. The mechanisms responsible for protein loss from plant culture media are not completely understood however, current indications are that multiple factors may be involved. Processes that have been proposed to affect foreign proteins in plant media include protein degradation due to protease activity [10, 17, 20, 38, 60, 65], protein instability due to defined or... 

PVP is a metabolically inert, water-soluble polymer with excellent protein stabilizing properties. As an example of the beneficial effect of PVP on foreign protein accumulation in plant tissue culture, data for growth and IgO, antibody levels in transgenic... 

The addition of PVP 360,000 at a concentration of 0.75 g L 1 has been reported to yield a 35-fold increase in the level of extracellular foreign protein in suspended plant cell cultures [66]. The effectiveness of PVP in stabilizing secreted proteins depends on both the polymer molecular weight and its concentration. Low-molecular-weight (10,000 and 40,000) PVP was found to be less effective than PVP 360,000... 

Although PVP has proven successful as a foreign protein stabilizer in several systems and has yielded substantial increases in product concentrations as discussed above, it has been found relatively ineffective in other plant tissue cultures producing foreign proteins [60, 65]. 

Modifying the properties of plant culture media, including increasing the osmolarity, reducing the effective concentration of selected heavy metals and altering the pH, has resulted in enhanced foreign protein accumulation or stability in some systems. 

Intact plants are also suitable for cost-effective production of recombinant pharmaceutical proteins providing products free of contaminations with endotoxins or human pathogens. Plants generally promote the proper fold of foreign proteins and post-translational modifications that are somehow similar to those of mammalian systems. Also, production of vaccine candidates in cereal seeds allows antigen protection from proteolysis which ensures their stability for a long period of time. 

Synthetic levothyroxine is the preparation of choice for thyroid replacement and suppression therapy because of its stability, content uniformity, low cost, lack of allergenic foreign protein, easy laboratory measurement of serum levels, and long half-life (7 days), which permits once-daily administration. In addition, T4 is converted to T3 intracellularly thus, administration of... 

As the biotechnology industry comes of age, and protein-based therapeutics become a practical reahty, the importance of protein-stabilizing technologies comes increasingly to the fore. The therapeutic efficacy of many of these biopharmaceuticals is impeded by the hosts natural immune defense system [23]. When a host encounters a foreign protein in its circulation, the hosts immune system initiates an immune response, which results in the production of protein-inactivating antibodies that dear the protein from the circulation. When the immune system develops an abihty to inactivate the protein, the therapy becomes ineffective. Hence, to counteract... 

Eed-batch feeding and induction policies that improve foreign protein synthesis and stability by avoiding stress responses. Biotechnol. Bioeng.,... 

Hoffman129 and Baier 13° have reviewed most of the hypotheses and mechanisms suggested for blood compatibility in general and for the role of protein adsorption in particular. The safest statement one can make is that protein adsorption is indeed important in the blood compatibility process, in the compatibility of soft contact lenses, in the stability and acceptance of intraocular lenses, in the soft tissue foreign body reaction 131), and in virtually all situations where solid surfaces come into contact with physiologic environments. 

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