COMPROBACIÓN DE HIPÓTESIS

1.2.3.1 Batch and fed-batch cultures

The large-scale production of foreign proteins in E. coli is influenced by a number of factors including strain selection, batch/fed-batch fermentation, temperature, composition of growth medium, the time and duration of induction and the nature of the protein being expressed. A range of antibody fragments have been produced in E. coli by fermentation and the use of both batch and high cell density fed-batch cultures has been investigated.

Batch fermentations generally result in low biomass concentrations and the titres of antibody fragments reported from such fermentations have varied from 40 mg L '1 (Berry et al., 1994) up to 450 mg L'1 (King et al, 1993). Fed-batch fermentations have been used to increase cell density and hence also titres. Carter and co-workers (1992a) achieved titres of 1-2 g L '1 of functional cell-associated protein in a 10L fermenter using a fed batch protocol. A mineral salts media supplemented with digested casein and controlled carbon source feeding were used and gave a final cell density of 120 to 150 O D 5 5 0 . Tight control of expression prior to induction was found

to be crucial to achieving high cell densities and thus high expression titres. Conditions were optimised for high titres of functional cell associated Fab’ and only low levels of Fab’ (<100 mg L '1) were found in the culture media.

A high cell density-fed batch fermentation of E. coli was also used by Horn et al., (1996) to produce functional dimeric miniantibodies. Titres o f 3 g L '1 were achieved using an optimised expression vector and high cell-density fermentation under non­ limited growth conditions, with levels of biomass reaching 40 g L '1 by the end of the fermentation. No periplasmic leakage or cell lysis was observed during the fermentation.

For both batch and fed-batch fermentations good fermentation development is required to minimise potential problems such as substrate inhibition, limited oxygen transfer capacity and the formation of growth-inhibitory by-products.

Production of acetate is a common problem with E. coli cultures growing in the presence of excess glucose. A high concentration of acetate (above 5 g L '1 at pH 7) reduces growth rate, maximum attainable cell density and hence also product yield (Han et al., 1992; Lee, 1996). Acetate formation depends on the strain, the medium and the specific growth rate and is generally greater in fed batch cultures than in batch cultures due to the extended culture period (Lee, 1996). One strategy for reducing acetate formation involves controlling the specific growth rate by limiting essential nutrients such as carbon and nitrogen (Korz et al., 1995; Yoon et al., 1993; Lee et al., 1989). Alternatively carbon sources which do not directly produce acetate such as glycerol may be used (Holms, 1986). Lowering the temperature of the culture from 37°C to 26-30°C can also be used to reduce nutrient uptake and growth rate, hence also reducing cellular oxygen demand, the formation of toxic byproducts and generation of heat. Lowering culture temperature has the additional advantage of increasing the titres of soluble product for some recombinant proteins (Cabilly, 1989; Takagi e ta l., 1988).

1.2.3.2 Development of growth media

The media selected for fermentations can affect both the yield of product and its location. Both complex and defined media have been used for antibody fermentations. Defined media are generally used to obtain high cell densities in fed batch culture as the nutrient concentrations are known and can be controlled during the fermentation (Pack et al., 1993; Horn et al., 1996). Complex media are more commonly used in batch fermentation and generally support higher specific growth rates (Shibui and Nagahari, 1992; Berry et al., 1994). However nutrients in complex media can vary in composition and quality and hence fermentations are less reproducible.

The composition of the growth media during the induction period can have an effect on the expression of recombinant proteins. Overexpression of a protein often imposes a metabolic drain on the cell’s energy, carbon and amino acid stores. This may result in reduced cell growth, increased plamsid instability and further physiological changes that reduce product yields. Provision of additional amino acids by supplementing the medium with casamino acids, peptone or yeast extract can significantly increase foreign protein expression and stability (Donovan et al., 1996).

1.2.3.3 Induction strategies using the lac promoter

The E. coli lac promoter is one of the most commonly used promoters for regulating the expression of recombinant genes in bacteria as it is well understood and has been extensively characterised (Donovan et al., 1996). A number of stronger promoters based on the lac system have been developed. These include the lacUV5 promoter which contains a mutation in the lac consensus sequence that increases promoter strength (Reznikoff and Abelson, 1980), and the tac promoter, a hybrid o f the tryptophan and lac promoters, which is reportedly 5-10 times stronger than the lacUV5 system (Amann et al., 1983).

The lac and associated promoters can be induced using isopropyl p-D-thiogalactoside (IPTG) or lactose. The tac promoter has the advantage of being lactose inducible while not being subject to catabolite repression (Donovan et al., 1996). IPTG is the more commonly used inducer because it is not metabolised by the cell, hence the levels of IPTG in the growth media remain constant after induction and the effects of altering the IPTG concentration on foreign protein expression can be easily assessed. The high cost of IPTG however may limit its use in large-scale processes. In addition IPTG may be toxic to humans and consequently its presence as a contaminant in the final purified protein destined for therapeutic use is undesirable. Lactose is much cheaper than IPTG, however because it is metabolised by the cell, optimising induction conditions for maximum foreign protein expression is a much more complex procedure.

The induction process is a critical stage in the production of foreign proteins in E. coli. Inducer concentration, temperature, point of induction and duration of the induction phase can all influence the titres of recombinant protein obtained.

The majority of work that assesses the effect of induction conditions on yields of recombinant proteins from the lac and associated promoters uses IPTG as the inducer. A wide range of IPTG concentrations have been reported (0.005 to 5 mmolL'1), however lmmol IPTG L '1 is most commonly used (Donovan et al., 1996). Shibui and Nagahari (1992) investigated the influence of IPTG concentration on secretion of a functional Fab in E. coli, and found that reducing the IPTG concentration from 1 mmol IPTG L'1 to 0.01-0.1 mmolL'1 resulted in a 2-10-fold increase in the yield of secreted Fab.

Shibui and Nagahari (1992) also investigated the effect of induction temperature on Fab secretion. Yields of Fab were significantly increased by growing cultures at 30°C instead of 37°C. The formation of inclusion bodies is also less prevalent at lower temperatures. For example, Cabilly (1989) reported a 10-fold increase in yield of soluble cytoplasmic Fab fragments when cultures were grown and induced at 21°C instead of 37°C.

It is likely that reduced inducer concentration and lower temperatures enhance functional protein formation by reducing rates at which the protein is formed. Lower expression rates reduce the concentration of the unfolded intermediate in the cell, which allows the polypeptide chains to interact via the correct folding pathways rather than those leading to aggregation.

The use of lactose as an inducer has also been assessed in a small number of studies. Lactose has been shown to be as effective as IPTG for inducing recombinant calf prochymosin and tyrosine phenol lyase using the tac promoter (Foor et al., 1993; Kapralek et al., 1991). For the expression of calf prochymosin induction of a batch culture with lactose produced greater yields than induction with 1 mmol IPTG L '1. High levels of product accumulated during late log and stationary phases with lactose

induction whereas recombinant protein was produced during the log phase of batch growth with IPTG.

A delayed response to induction with lactose was also observed in the production of hoof and mouth disease viral protein 1 under the control of the T7 phage promoter/polymerase system controlled by the lac promoter (Neubauer and Hofmann, 1994). For this system the optimal time for lactose induction was found to be just before the glucose was depleted from the medium in late log phase; the addition of lactose one hour later in the stationary phase produced poor yields of the target protein.