A number of parameters must be considered for the development of an efficient expression system in E.coli

•The stability of the mRNA, •The efficiency of mRNA translation •The accuracy of amino acid incorporation•Correct folding •Proteolysis of product •Protein transport

For the expression of a recombinant proteins in E. coli, an expression vector is required which should contain, apart from the gene of interest, an origin of replication, a gene that confers resistance to some antibiotic, a promoter and regulators of transcription and translation.

Promoters The promoter should have certain characteristics to render its suitability for high level protein expression.• It should be transcriptionaly strong.•It should be tightly regulated.•It should be induced in an cost effective manner (Chemical, growth condition or growth nutrients).promoters from the lac operon and the tryptophan (trp) biosynthetic operon as well as phage promoters such as the λPL promoter (and the φ10 promoter from phage T7T

The use of IPTG for large-scale production of human therapeutic proteins is undesirable because of its toxicity and its detrimental effects to the host physiology . Other promoters like, pH inducible promoter , oxygen-regulated promoter , salt inducible proUp promoter and araB promoter (arabinose inducible) are providing additional options for high level gene expression.

Upstream elements

The flanking DNA regions of core promoters play an important role in determining transcription efficiency. Upstream (UP) elements located at 5’ of the –35 hexamer in most bacterial promoters are A+T rich sequences that increase transcription by interacting with the α subunit of RNA polymerase.

The positioning of highly active upstream sequences upstream of well repressed promoters may increase their strength to a level comparable with phage promoters, but without the drawbacks associated with phage polymerase expression.

mRNA stability

The prokaryotic mRNAs are unstable with a typical half-lives ranging between 30 s to 20 min. The major enzymes involved in mRNA degradation are two 3’-5’ exonucleases {RNase II and polynucleotide phosphorylase (PNPase)} and the endonuclease Rnase.

The stable secondary structures present in the 5’ UTR of certain transcripts as well as in 3’ rho-independent terminators can both increase mRNA stability. The stabilizing effect conferred by untranslated 5’ hairpins was first demonstrated in the case of the long-lived ompA mRNA. Fusion of the ompA 5’ UTR to a variety of heterologous mRNAs significantly increased transcript half-life, presumably by interfering with RNase E binding.

Shine-Dalgarno sequenceInitiation of translation of E. coli mRNAs requires a Shine-Dalgarno (SD) sequence complementary to the 3’ end of the 16S rRNA which has the consensus sequence 5’-UAAGGAGG-3’, followed by an initiation codon, which is most commonly AUG.

This also means that stable mRNA secondary structures encompassing the SD sequence and/or the initiation codon can dramatically reduce gene expression by interfering with ribosome binding. This problem can be circumvented by increasing the homology of SD regions to the consensus and by raising the number of A residues in the initiation region through site-directed mutagenesis.

Codon biasness: Prokaryotes and eukaryotes have major difference in codon usage, which can have significant effects on heterologous protein expression. The arginine codons AGA and AGG are rarely found in E. coli genes, whereas they are common in S. cerevisiae and eukaryotes. The presence of such codons in cloned genes affects protein accumulation levels, mRNA and plasmid stability and in extreme cases inhibit protein synthesis and cell growth .

An important but much less obvious effect of AGA codons, is primary structure changes due to the misincorporation of lysine for arginine, particularly when cells are grown in minimal medium (Forman et al., 1998). Fortunately, these problems can usually be addressed by using site-directed mutagenesis which is used to replace rare arginine codons by the E. coli-preferred CGC codon, or by co-overexpressing the argU (dnaY) gene which encodes the tRNA for arginine.

E. Coli as an Expression host

E. coli has been successfully used for the expression of recombinant proteins because of its well characterized genetics and growth conditions.

By using well-established cultivation strategies of high cell density cultivation, a number of proteins have been produced at gram levels .

E. coli’s capacity to accumulate foreign proteins to more than 20 % of its total cellular protein have made this organism the most widely used prokaryotic system for recombinant protein production. Although E. coli is a widely used expression system it has some disadvantages.

Sometimes, the overexpressed protein tends to accumulate in the bacteria as an insoluble intracellular product (inclusion bodies) which are misfolded and often very difficult to refold to the correct native state.

The contamination of endotoxins in E. coli derived products is a major problem and its consequent removal is mandatory as they are ubiquitous pathogenic molecules.

The proteins produced in E. coli are not glycosylated. Glycosylation even when it is not necessary for biological activity, often increases the stability of proteins and influences reaction kinetics, solubility, serum half-life, thermal stability, immunogeneticity and receptor binding

Expression strategy Advantages Disadvantages

Cytoplasmic expression (Inclusion bodies)

Inclusion bodies are easy to purify.Protection from degradation by proteases.Beneficial for toxic protein expression.High production yields are usually obtained.

Normally no authentic N-terminus.Refolding required for active protein. Poor refolding yields High cost of solubilization.

Cytoplasmic expression (Soluble expression)

No need of solubilization and refolding.Usually active protein

Disulfide bond formation usually not possibleHigh level of intracellular product can be harmful to host cellsComplex and costly purificationProtein proteolysis might occur.

Periplasmic expression Disulfide bond formation possibleReduced level of contaminantsPossible to obtain authentic N-terminus

Secretion to periplasm not always possiblePeriplasmic protease can cause proteolysisNo large-scale procedure possible for selective release of heterologous protein from periplasm.

Extracellular production Disulfide bond formation possibleNo need for cell lysisPossible to obtain authentic N-terminusProteolysis of recombinant protein avoided.

Secretion to the medium usually not possible.Dilution of the product

Production mode•Batch •Fed batch •Continuous cultures

Factors affecting high cell density cultivation

Though HCDC fed-batch cultivation is a popular cultivation strategy, it is associated with certain limiting factors. The problems arising due to high cell density are accumulation of by-product, limitation of dissolved oxygen concentration, increase of temperature, poor mixing and degradation of product.

Problem of acetate production in E. coli

One of the major technical challenges in recombinant protein production is to achieve high expression levels of the cloned gene in the individual cell (specific product formation rate) and also high cell density. Unfortunately, under the demanding conditions of HCDC, the amount of acetate accumulation in the reactor increases enormously often to a level that has a detrimental effect on cell health and hence protein yields. The presence of excess glucose and oxygen limiting conditions during cultivation leads to accumulation of acetate in the culture .

Acetate is produced when the carbon flux into the central metabolic pathways (TCA cycle) exceeds the biosynthetic demands and the capacity for energy generation within the cell. Accumulation of > 0.5 g/l of acetate reduces growth rate, biomass yields and maximum attainable cell densities in high cell density cultures (as well as specific product formation rate

Strategies to solve the problem of acetic cid productionVarious operational strategies have been proposed and tested in the past to reduce the extent of acetate accumulation. Most of these approaches fall into one of the following categories.(a) Selection of a production strain with low acetate.(b) Adjustment of the medium feed rate in accordance with the oxygen transfer capacity of the reactor).(c) Use of oxygen enriched air or pure oxygen for aeration.(d) In situ removal of acetate by perfusion systems.(e) Construction of mutant strains with reduced acetate formation(f) Use of alternative substrates, which reduce the formation of acetate, such as glycerol.

b) Dissolved oxygen limitation

The dissolved oxygen concentration becomes limiting in high cell density cultivation owing to its low solubility. Oxygen starvation has critical effects on cell health that some times leads to cell lysis and protease activation. Increasing the aeration rate or agitation speed can increase the dissolved oxygen concentration.

In HCDC the supply of pure oxygen has also been used. However pure oxygen is expensive and higher concentration of oxygen is toxic to cells.

As oxygen consumption increases with the growth rate, the oxygen demand of cells can be reduced by lowering the growth rate. These strategies can be combined to achieve a high cell density of various E. coli cultures

c). Cultivation Temperature

Temperature is an important variable that can be used to control cell metabolism. By lowering the culture temperature from 37 oC to 26-30 oC, nutrient uptake and growth rate can be reduced, thus reducing the formation of toxic by-products and the generation of metabolic heat.

Lowering of cultivation temperature also reduces cellular oxygen demand, which enables higher cell-densities to be obtained without the need of pure oxygen.

Furthermore is possible to reduce the formation of inclusion bodies for some proteins by growing recombinant cells at a lower temperature

Other problems like mixing efficiencies carbon dioxide evolution rate and heat generation in large-scale fermentation are also problems that arise during HCDC.

Robust feeding strategies and process optimization

Fed-batch cultivation is usually done in two phases; a). a batch phase cultivation with a maximum specific growth rate (μ = μmax) and a fed-batch phase with a reduced specific growth rate (μ< μmax).

The reduced growth rate is necessary to prevent the accumulation of inhibitory by-products. The reduced growth rate also helps in obtaining higher product formation rates.

Substrate limited fed-batch strategies are common for high cell density cultivation. There are two feeding strategies for the control of the nutrient feed; a). Open loop control and the closed loop feed back control.

Open loop feeding

Open-loop fed-batch strategies use a certain predetermined feeding profile. After a batch phase without feeding, an exponentially increasing feeding rate has to be applied in order to maintain a more or less constant specific growth rate.

The feeding strategy not only affects the maximal achievable cell density, but also the cell productivity .Feeding strategies like feeding at constant, a step wise increase of feeding rate and an exponential feeding comes under open loop feeding control (without feed back)

In constant feeding the specific growth rate declines continuously with cultivation time.

In an exponential feeding strategy a concentrated feed is fed at an exponential rate to maintain a predetermined specific growth rate of the culture.

Closed loop (Feedback) feeding

For closed-loop feedback control of physicochemical and environmental parameters, such as temperature, pressure, pH, foam, stirrer speed etc., well-known closed-loop controllers (PID-proportional-integral-differential controller, switching controllers and others) are well established in bioprocess optimization. The use of DO Stat, pH Stat and Carbon evolution rate (CER) are common closed loop feeding strategies having wide application in high cell density cultivation.

DO stat feeding

Dissolved oxygen (DO) is an important parameter to measure the cellular health. Critically low DO concentrations can effect the recombinant protein production in fed-batch cultures. Hence a feeding strategy to maintain a constant dissolved oxygen concentration by feeding concentrated feed is used for HCDC in fed batch culture.

This strategy is based on the finding that the DO value sharply increases when the growth limiting substrate is consumed. Therefore addition of concentrated feed is done to maintain the preset value of dissolved oxygen concentration This feeding strategy has greater impact when defined feed medium is used for cultivation.

When complex carbon and nitrogen substrate such as yeast extract, peptone and tryptone are used together with the carbohydrate substrate, the DO change is not as sharp when the carbon source is depleted, as the cells continue to utilize the complex substrates

pH Stat Feeding

The cultivation pH is an important parameter for recombinant protein production at large scale. High culture pH is known to induce host proteases which some times leads to recombinant protein degradation.

The pH value begins to rise when the carbon substrate is exhausted; this increase in pH is mainly due to the increase in the concentration of ammonium ions excreted by cells (Suzuki et al., 1990). The pH Stat method is more suitable, when semi-defined or complex media is used for cell growth. The pH is kept constant by controlling the feed rate.

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