Total soluble protein

The gene for the dehydratase was expressed in E. coli under lac promoter, and an expression plasmid pOxD 90F was constructed. The transformant was cultivated under optimal condition at 30° C when much of the enzyme was expressed in a soluble form with more than thousand and several hundred times than the wild-type strain per culture (up to more than 50% of the total soluble protein of the... 

Allen et al. (2007) produced puffed snack foods with com starch and pregelatinized waxy starch, WPC and instantized WPC, and protein concentrations of 16%, 32%, and 40% and showed that the air cell size, extru-date expansion ratio, and water solubility index decreased proportionally as protein and com starch levels increased. Protein concentration significantly affected total soluble protein, water absorption index, and water-soluble carbohydrate. A covalent complex between amylase and protein formed in the presence of cornstarch, but protein-protein interactions appeared with the presence of low levels of pregelatinized waxy starch. 

B. megaterium, in contrast to B. subtilis, has low protease activity. It is able to grow on a wide variety of substrates. Plasmids with xylose-inducible promoter are most frequently used for high-level expression [41]. It was successfully used to express toxins and other difficult proteins where the proteins were intact after secretion into the media. Yields of about 30% of total soluble protein were obtained in some cases [42]. 

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]. 

Fig. 1.1 Promoter activity in alfalfa leaves. Accumulation ofp-glucuroni-dase achieved in transgenic alfalfa leaves expressing the gusA gene under the control of CaMV 35S and alfalfa promoters. %TSP, percentage of total soluble proteins.

Fig. 1.2 Protein blot analysis of human therapeutic protease inhibitor (HTPI) produced in alfalfa cell cultures using different promoters and subcellular targeting peptides as shown. Equal amounts of total soluble proteins from cell cultures were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinyldifluoride (PVDF) membrane. Monoclonal anti-HTPI IgGs were used for detection.

Fig. 1.4 Protein blot analysis of C5-1 assembly in agroinfiltrated alfalfa leaves. Total leaf soluble proteins, extracted 4 days after infiltration were separated by SDS-PAGE under non-reducing conditions and blotted onto a PVDF membrane. Polyclonal antimouse IgGs were used for detection. Purified C5-1 was mixed with total soluble proteins from control infiltrated alfalfa leaves and loaded as a standard.

CaMV = cauliflower mosaic virus DMSO = dimethylsulfoxide PVP = polyvinylpyrrolidone TSP = total soluble protein U = unit... 

Fig. 3.2 Rapeseeds were germinated from 12to 168 h in airlift tank. The total soluble proteins were extracted and separated by 15% SDS-PAGE. The gel was stained with Coomassie blue. Between 36 and 60 h, the degradation of storage proteins and the de novo synthesis of Rubisco is clearly visible.

Currently, we can produce 1.5 g of GUS protein and 0.5 g of HSA per kg of seeds. These levels correspond to 13.5kg and 4.5 kg production yields per year, respectively. Expression levels are indicated as a weight per weight of starting material (dry seeds) because the sprout fresh weight and total soluble protein content varies depending on cultivation time and conditions. 

Fig. 8.3 Expression of IGF-1 in transgenic chloroplasts after continuous light exposure for 13 days. IGF-1 expression is shown as a percentage of the total soluble protein. IGF-1 n is the native gene and IGF-1 s is the chloroplast codon-optimized gene.

Fig. 8.4 Coomassie-stained SDS-polyacrylamide gel showing chloroplast transgenic lines expressing IFNa2b. Lanes 1 and 2 Total soluble protein (TSP) Lanes PH, 3 and 4 Total protein (TP).

Fig. 8.7 CTB-GM1-ganglioside binding ELISA assay. Plates, coated first with GMrganglioside and bovine serum albumin (BSA), respectively, were irrigated with total soluble plant protein from chloroplast transgenic lines (3 and 7) and 300 ng of purified bacterial CTB. The absorbance of the GM1-ganglioside-CTB-antibody complex in each case was measured at 405 nm. Total soluble protein from untransformed plants was used as the negative control.

Seed-specific production of LTB by Streatfield et al. [28] resulted in expression levels of up to 1.8% total soluble protein (TSP), and two separate maize breeding programs have increased antigen production by fivefold [28] and tenfold [27]. The investigations of Chikwamba et al. [27] regarding the expression of LTB in maize are among the first to include the use of particle bombardment transformation for the production of plant-derived vaccines. 

Tab. 15.1 Therapeutic antibodies produced in transgenic plants. See Fig. 15.1 for structures of the different antibodies and antibody fragments presented in the second column. Abbreviations TSP, total soluble protein FLW, fresh leafweight MSP, murine signal peptide SP, signal peptide KDEL, ER retention signal. In the column Expression + and - means respectively that the antibody was or was not expressed. 

A wide range of mammalian proteins have been expressed in S. pombe. In a successful example, the human lipocortin I comprised 50% of the total soluble proteins in yeast cells and showed high activity, indicating that the post-translational modifications were mammalian-like [104]. Membrane proteins including cytochrome P450 were expressed at ten times the levels of those in other yeast systems [105]. Also, GPCRs have been expressed in S. pombe, where the human dopamine D2 receptor was correctly inserted into the yeast cell membrane and demonstrated expression levels three times those of S. cerevisiae [106]. 

The amount of nitrile hydratase in the cell-free extracts accounts for more that 50% of the total soluble protein. 

The team from Icongenetics used the deconstructed virus system to attain protein yields of up to 80% of total soluble protein or 5 g/kg of freshwater biomass, a process that only takes 3-14 days. Development of a scaled-up version of this expression system in transgenic plants by controlling the activation of an encrypted version of a replicon present in the plant chromosome of a production host is currently under investigation. One enormous advantage to the system would be the lack of requirement of systemic movement of the virus. 

The amount of vaccine or therapeutic protein produced in a plant can reach industrial-scale levels. The typical yield of biopharmaceuticals produced in a plant-based system is 0.1%-1.0% of total soluble protein (Twyman et al., 2003). This value is competitive with other expression systems therefore, a plant expression platform for biopharmaceuticals is economically viable. As an example, one bushel of maize can produce as much avidin as one ton of chicken eggs but at 0.5% the cost. 

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