Secretion of Foreign Proteins

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]. 

Alterations to the proteins and pre-proteins expressed by cultured plant cells have been used to facilitate product recovery. A leader sequence is required for foreign protein secretion from plant cells into the apoplast and then into the culture medium. As indicated in Table 2.1, plant, mammalian and viral sequences have been employed to achieve the entry of transgenic proteins into the bulk-flow pathway in plant cultures. 

A vector that facilitates high-level protein expression in plant tissue culture, particularly a transient expression system that could be applied to existing wild-type cultures, would be advantageous for in vitro foreign protein production. However, such a system has not yet been developed. The success of this approach depends in part on whether appropriate levels of viral infection, replication and transmission can be established within tissue culture systems. 

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 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. 

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Deng, T., Noel, J. P., and Tsai, M.-D. (1990). A novel expression vector for high-level synthesis and secretion of foreign proteins in Escherichia coli Overproduction of bovine pancreatic phospholipase A2. Gene 93, 229-234. 

To promote the secretion of foreign proteins, the Tat-dependent signal sequence torA from E. coli, connected to the C. thermocellum endoglucanase gene,... 

The simplicity of plant culture media is considered an advantage for foreign protein production in tissue culture systems. However, as a mixture of salts and sugar containing several heavy metals but negligible protein (except for any protein secreted... 

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... 

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... 

Continuous or periodic harvesting of secreted foreign proteins from plant culture media could be used to increase product yields by removing active protein before it can be degraded or otherwise lost from the culture. 

It is unclear why certain foreign proteins can also stimulate the B-cells to secrete IgE antibodies, to result in allergy or hypersensitivity. The terms are used interchangeably, although the latter is usually restricted to milder forms of the response. The term anaphylaxis is used to describe the severe response (Box 17.4). Both reactions arise in genetically susceptible individuals and they are precipitated by exposure to environmental antigens such as pollen, some organic compounds, tobacco smoke, animal hairs or even components of some common foods such as milk and cereals. 

A major goal in recombinant DNA technology is the production of useful foreign proteins by bacteria, yeast, or other cultured cells. Protein synthesis depends upon both transcription and translation of the cloned genes and may also involve secretion of proteins from the host cells. The first step, transcription, is controlled to a major extent by the structures of promoters and other control elements in the DNA (Chapter 28). Since eukaryotic promoters often function poorly in bacteria, it is customary to put the cloned gene under the control of a strong bacterial or viral X promoter. The latter include the X promoter PL (Fig. 28-8) and the lac (Fig. 28-2) and trp promoters of E. coli. These are all available in cloning vehicles. 

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