Protein accumulation

Urea Pharmacokinetics. Pharmacokinetics summarizes the relationships between solute generation, solute removal, and concentration in a patient s blood stream. In the context of hemodialysis, this analysis is most readily appHed to urea, which has, as a consequence, become a surrogate for other uremic toxins in the quantitation of therapy and in attempts to describe its adequacy. In the simplest case, a patient is assumed to have no residual renal function. Urea is generated from the breakdown of dietary protein, accumulates in a single pool equivalent to the patient s fluid volume, and is removed uniformly from that pool during hemodialysis. A mass balance around the patient yields the following differential equation ... 

Unfolded protein response, when mis-folded proteins accumulate in the ER, signal transduction pathways are activated that increase the biosynthetic capacity and decrease the biosynthetic burden of the ER. 

The 26 kDa protein synthesised by salt-adapted tobacco cells has been further characterised (Singh et al., 1987a). The protein makes up approximately 12% of the total cellular protein and has been resolved into two forms. These two forms have been designated osmotin 1 and osmotin II and occur in a 2 3 ratio. The forms are distinct with osmotin I soluble in an aqueous phase and osmotin II soluble in detergent. The proteins accumulate as inclusion bodies in the vacuole and are only sparsely distributed in the cytoplasm. 

Mutation of the dihydrolipoate reductase component impairs decarboxylation of branched-chain a-keto acids, of pyruvate, and of a-ketoglutarate. In intermittent branched-chain ketonuria, the a-keto acid decarboxylase retains some activity, and symptoms occur later in life. The impaired enzyme in isovaleric acidemia is isovaleryl-CoA dehydrogenase (reaction 3, Figure 30-19). Vomiting, acidosis, and coma follow ingestion of excess protein. Accumulated... 

Toxic metabolites such as lactic acid and denatured proteins accumulate. The pH fells with accumulation of H" ions (Green and Pe, 1979). 

The first hurdle encountered during the development of alfalfa as a recombinant protein production system was the relative inefficiency of the available expression cassettes. A study in which a tomato proteinase inhibitor I transgene was expressed in tobacco and alfalfa under the control of the cauliflower mosaic virus (CaMV) 35S promoter showed that 3-4 times more protein accumulated in tobacco leaves compared to alfalfa leaves [5]. Despite the low efficiency of the CaMV 35S promoter in alfalfa, bio-pharmaceutical production using this system has been reported in the scientific literature. Such reports include expression of the foot and mouth disease virus antigen [6], an enzyme to improve phosphorus utilization [7] and the anti-human IgG C5-1 [8]. In this last work, the C5-1 antibody accumulated to 1% total soluble protein [8]. 

Strategies for Improving Foreign Protein Accumulation and Product Recovery in Plant Tissue Culture... 

Compared with whole plants, there has been limited development of foreign protein expression systems specifically for use in tissue culture. Some modifications of expression constructs have resulted in improved protein accumulation or have allowed simplified protein recovery. However, in general, modified expression systems have been tested only in a restricted number of cases and have not resulted in the large increases in product yield required for plant cultures to compete with other foreign protein production vehicles. Transient expression techniques, for example using viral vectors, that have been developed for use in whole plants have not yet been applied in plant tissue culture. 

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]. 

Proteins produced in plant cells can remain within the cell or are secreted into the apoplast via the bulk transport (secretory) pathway. In whole plants, because levels of protein accumulated intracellularly, e. g. using the KDEL sequence to ensure retention in the endoplasmic reticulum, are often higher than when the product is secreted [58], foreign proteins are generally not directed for secretion. However, as protein purification from plant biomass is potentially much more difficult and expensive than protein recovery from culture medium, protein secretion is considered an advantage in tissue culture systems. For economic harvesting from the medium, the protein should be stable once secreted and should accumulate to high levels in the extracellular environment. 

The results of empirical studies carried out to test the effects of medium additives on foreign protein accumulation in plant tissue culture are summarized below. 

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. 

When sufficiently high levels of expression and protein accumulation are achieved, efficient downstream processing protocols must be developed to insure product quality and the economic feasibility of production. As the demand for safe, recombinant pharmaceutical proteins continues to expand, the market potential of plant-produced recombinant proteins is considerable. Molecular farming can produce recombinant proteins at a lower cost than traditional expression systems based on microbial or animal cell culture, and without the risk of contamination with human pathogens. 

In order to make molecular farming commercially profitable, recombinant proteins must be produced at a sufficiently high yield and in an active form. It has become clear that, for high-level protein accumulation, the stability of transgene expression can be as important as the expression level itself. The quantity of protein is determined by the rate of protein synthesis, assembly as well as proteolytic degradation [83]. 

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