Proteins alfalfa

Respiratory syncytial virus (RSV) G and F proteins Alfalfa mosaic virus in tobacco leaf High levels of serum antibodies specific for RSV-G. Immunogenic in mice when delivered parenterally. Protective against challenge with RSV Long strain. 30... 

Human immunodeficiency virus (HIV) type 1 gpl20 (V3 loop) protein Alfalfa mosaic virus in tobacco leaf Elicited specific virus-neutralizing antibodies in mice when delivered parenterally. 16... 

Wang, J.C. and Kinsella, J.E. 1976. Funtional properties of novel proteins Alfalfa leaf protein. J. Food Sci. 41 286-292. 

Adrenodoxin Apoadrenodoxin Testis iron protein Alfalfa ferredoxin (23) maximum cm-1M-1 maximum cm 1M 1 maximum cm-1M 1 maximum cm 1M 1... 

Smith, T.K. (1980b). Effect of dietary protein, alfalfa and zeolite on excretory patterns of 5, 5 , 7 , 7 -[%] zearalenone in rats. Can. Physiol. Pharmacol. 58, 1251-1255. 

Legume forages, such as alfalfa or clover, are considered high quaHty, readily available protein sources. Animal sources of supplemental protein include meat and bone meal blood meal, 80% CP fish meal other marine products and hydroly2ed feathermeal, 85—90% CP. Additionally, synthetic amino acids are available commercially. Several sources (3,9,19) provide information about the protein or amino acid composition of feedstuffs. 

Table 11 presents data on the protein quaUty of a variety of LPC products obtained from rat-feeding studies. Typical protein efficiency ratio (PER) values for LPCs derived from alfalfa range from 1.41 without supplementation to 2.57 with 0.4% methionine added casein can be adjusted to a PER of 2.50 (98,100). Biological values (BV) of mixtures of LPCs, such as barley and rye grass or soybean and alfalfa, maybe higher than either LPC alone. The effect has been attributed to the enhanced biological availabihty of lysine in these mixtures (99). 

The 1993 market for LPC-type products in the United States was for dried alfalfa meal for animal feed. This product is sold for both protein and carotenoid content. The USDA Pro-Xan product attempts to obtain improved xanthophyU contents for use in egg-laying rations in addition to protein contents. The limitations to commercial development of LPC products for human food use are high capital costs as compared with the low yields of protein, seasonal availabihty of raw materials, and the need in the United States for FDA approval of the products. 

This chapter provides an overview of the tools that have been developed and optimized specifically for the production of pharmaceuticals in alfalfa, with the emphasis on recent technological breakthroughs. The ability of alfalfa leaves to produce complex recombinant proteins of pharmaceutical interest is discussed and illustrated with recent data obtained in our laboratories. Data are presented concerning the production and characterization of alfalfa-derived C5-1, a diagnostic anti-human... 

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.

In order to reduce the time required to confirm the accumulation of a given recombinant protein, we have developed a cell culture system in which transgenic alfalfa callus material produced at the proliferation step of Agrobacterium-based transformation is used to initiate cell cultures. These cell suspensions can be subcultured to sustain batch production of modest protein amounts. The protein blot shown in Fig. 1.2 demonstrates our ability to detect a recombinant protein in total... 

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. 1.3 Prediction of the most appropriate subcellular targeting strategies by agroinfiltration. The levels of an industrial enzyme (IE) are shown in agroinfiltrated and transgenic alfalfa leaves using different subcellular targeting peptides. Equal amounts of total soluble leaf proteins were separated by SDS-PAGE and blotted onto a PVDF membrane. Polyclonal anti-IE 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.

The production of C5-1 by co-infiltration illustrates the impressive capacity of this method. Results presented in Fig. 1.4 show that the production of C5-1 in detached alfalfa leaves was validated within 5 days from infiltration. In these experiments, different bacteria bearing the light- and the heavy-chain constructs were used to infect the cells. Most of the infected cells were occupied by both strains, and a protein corresponding to fully assembled C5-1 was detected in the infiltrated leaf extract This result demonstrates the potential of agroinfiltration for testing the adequate expression and assembly of complex proteins in alfalfa leaves using different Agrobacterium strains. 

When recombinant proteins are produced in a heterologous system, there may potentially be differences between the final product and the natural molecule. Hence, for each new protein produced in alfalfa, a thorough analysis of the processing, folding, assembly and post-translational modification is conducted to ensure the conformity of the purified molecules. This section describes the analysis of alfalfa-derived... 

Fig.1.5 MALDI-TOF mass spectra of purified alfalfa-derived C5-1 using (a) h uman IgG or (b) protein A. (c) Hybridoma-derived C5-1 as control. Used with permission from Ref 18.

In the tight of the results presented above, we conclude that alfalfa offers a suitable system for the high-yield production of correctly assembled complex proteins, including multimeric glycoproteins. The post-translational capacities of alfalfa indicate that this system is one of the best-suited for the production of molecules for therapeutic and diagnostic applications. 

M.-A. DAoust, U. Busse, M. Martel et al., Alfalfa An efficient bioreactor for continuous recombinant protein production, in Molecular Farming of Plants and Animals for Human and Veterinary Medicine. 2003, eds. L. Erickson,... 

The amino acid composition of storage proteins differs from that of the complete sprout [12, 13]. At least in the case of oilseed rape, alfalfa (Medicago sativa L.) and Camelina sativa, amino acids in the sprout are used mainly, either directly or indirectly, for the synthesis of the Rubisco proteins. Computer analysis shows that the amino acid composition of cruciferin and napin is completely different to the amino acid composition of Rubisco. This indicates that amino acids released from the seed storage proteins must be converted into other amino acids prior to Rubisco synthesis. 

In the quest to find other plants that are suitable as bioreactors, various monocoty-ledonous and dicotyledonous species have been tested. These include corn [16], rice and wheat [17], alfalfa [18], potato [19, 20], oilseed rape [21], pea [22], tomato [23] and soybean [24]. The major advantage of cereal crops is that recombinant proteins can be directed to accumulate in seeds, which are evolutionar specialized for storage and thus protect proteins from proteolytic degradation. Recombinant proteins are reported to remain stable in seeds for up to five months at room temperature [17] and for at least three years at refrigerator temperature without significant loss of activity [25]. In addition, the seed proteome is less complex than the leaf proteome, which makes purification quicker and more economical [26]. 

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