Choice of an Expression System

Expression of genes at a regularly low level, resulting in a low level of product, occurs in every organism. For recombinant proteins, overexpression which leads to considerably enhanced amounts of the recombinant protein is desired. Success of an attempt at recombinant overexpression cannot be predicted as of yet and depends on many factors, such as the protein to be expressed, host, vector, promoter, culture conditions, and, last but not least, the experience of the investigator. 

The two most important factors in the choice of an expression system should be (i) economy and efficiency, and (ii) the greatest possible closeness of the host to the DNA source if posttranslational modifications are required. However, in the case of eukaryotic genes, compromises are frequently necessary. The need for a close relationship often rules out overexpression, because often no efficient high-yield expression system exists among related eukaryotes one of the best known systems employs CHO (Chinese hamster ovary) cells. 

A few problems during overexpression, which have been dealt with, are mentioned here. Recombinant proteins can be toxic to the host then overexpression results in low expression rates, lysis of the cells, or proteolysis of the recombinant protein. Options other than changing the host, which often is impossible, include regulation of culture conditions, change of inductors, or secretion of the recombinant protein. Proteolysis can be avoided by 

Since constitutive expression in high yields negatively influences cell metabolism with consequences up to and including cell death, the commonly used expression systems are inducible. The goal of inducible overexpression is a yield of soluble target protein between 5 and 50%, typically about 20%, of total cell protein. If expression as soluble protein is deemed impossible, alternatives are (i) expression as inclusion bodies (insoluble protein fraction), (ii) expression as a fusion protein, or (iii) secretion into the medium. For a good ratio of soluble to insoluble recombinant protein (the ratio is hardly ever infinity), expression criteria have to be optimized. 

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This example clearly illustrates not only the range of clinical manifestations with respect to antibody formation to drug therapy but also how the choice of an expression system can affect the hnal product. In this example, the expression system was responsible for the adverse events reported. This finding is certainly clinically relevant considering the homologous product, sargramostim, has been on the U.S. market for quite some time. 

In summary, the criteria for the choice of an expression system in pharmaceutical production [82] are the existing expertise, the available physiological and genetic know-how and tools, the patent situation, and to avoid delays of product launch and commercialization, regulatory aspects like the acceptance by the approving authorities. 

If, instead of making an arbiPary choice of the coordinate system, we choose more wisely, the ellipse can be expressed more simply, without cross temis [Eq. 2-43)]... 

Therapeutic proteins have also been successfully expressed in the urine and seminal fluid of various transgenic animals. Again, issues of sample collection, volume of collected fluid and the appropriateness of these systems render unlikely their industrial-scale adoption. One system that does show industrial promise, however, is the targeted production of recombinant proteins in the egg white of transgenic birds. Targeted production is achieved by choice of an appropriate... 

Once an expression system is chosen, it is beneficial to optimize the variables of the system to maximize production. If a bacterial system is snfficient, a choice has to be made between vectors for intracellnlar production and secretion. This choice is made on considerations of downstream pnrification and generation of bioactivity. Intracellular expression resnlts in high yields, but often leads to the protein s aggregation into inclnsion bodies (Georgiou and Bowden, 1991 Papontsakis, 1991). 

In this section, the development of a few of the currently approved protein drugs is examined with emphasis on the choice of expression system, method of purification, and other factors that have had an influence on their commercialization. At least one drug produced through each of the expression systems is considered to illustrate the rationale for the choice and the influence of this choice on production, purification, and regulatory aspects. 

Membranes are particularly suited for bioprocesses involving the cultivation of microorganisms or cells as biocatalysts, in which the product of interest is produced extracellularly. Such processes are becoming increasingly attractive when compared to those in which the products accumulate intracellularly. Some of the reasons for this include the use of novel expression systems which favor higher product concentrations, and the ease of purification as compared to an intracellular bioproduct route. One of the drawbacks remains that extracellular protein products are produced in dilute concentration. Extracellular-product based-processes require cell separation, product recovery and concentration. The use of ultrafiltration and microfiltration membranes has become a method of choice in such process schemes. 

The structural complexity of a specific bio-pharmaceutical protein, together with the amount required, determine the choice of an appropriate expression system. While antibody fragments can easily be produced in yeasts, fungi, or E. coli cells, the expression of whole antibodies still requires complex mammalian cell culture processes. 

We conclude this section by discussing an expression for the excess chemical potential in temrs of the pair correlation fimction and a parameter X, which couples the interactions of one particle with the rest. The idea of a coupling parameter was mtrodiiced by Onsager [20] and Kirkwood [Hj. The choice of X depends on the system considered. In an electrolyte solution it could be the charge, but in general it is some variable that characterizes the pair potential. The potential energy of the system... 

Outlined below are the steps required for of a X T.E calciilation of vapor-phase composition and pressure, given the liquid-phase composition and temperature. A choice must be made of an equation of state. Only the Soave/Redlich/Kwong and Peng/Robinson equations, as represented by Eqs. (4-230) and (4-231), are considered here. These two equations usually give comparable results. A choice must also be made of a two-parameter correlating expression to represent the liquid-phase composition dependence of for each pq binaiy. The Wilson, NRTL (with a fixed), and UNIQUAC equations are of general applicabihty for binary systems, the Margules and van Laar equations may also be used. The equation selected depends on evidence of its suitability to the particular system treated. Reasonable estimates of the parameters in the equation must also be known at the temperature of interest. These parameters are directly related to infinite-dilution values of the activity coefficients for each pq binaiy. 

The expression of the models in different modes of representation occurred during the whole process and, as has been previously commented upon, exerted an essential role in the development of students knowledge. This was particularly relevant for those students who could understand the relevance of the choice of a given code and level of representation in order to better express the mental model previously produced. In several activities, students were asked to propose a concrete model for a specific system. This was shown to be essential for the development of students ideas because, from the concrete models, they could produce simulations of the chemical process and think about details related to the mechaiusm of the chemical reactions (such as the directions of the necessary collisions between the molecules, something that they had not studied before). 

Within each species, individual promoters resulted in distinct, tissue-dependent accumulation patterns. The cauliflower mosaic virus (CaMV) 35S promoter, for example, led to high-level accumulation in callus and leaves whereas the maize ubiqui-tin-1 promoter was the best choice for producing recombinant proteins in cereal seeds even though it is not in itself seed-specific [23]. The lack of such comparative studies for proteins other than rAbs makes it difficult to generalize an optimal expression strategy for all proteins. Tables 7.1 and 7.2 list recombinant proteins expressed in plants and provide details of the production system, promoters and other regulatory elements used in each case. 

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