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.

Other promoters like. • It should be transcriptionaly strong. 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 .Promoters The promoter should have certain characteristics to render its suitability for high level protein expression. salt inducible proUp promoter and araB promoter (arabinose inducible) are providing additional options for high level gene expression. growth condition or growth nutrients). oxygenregulated promoter . . pH inducible promoter . •It should be tightly regulated. •It should be induced in an cost effective manner (Chemical.

The positioning of highly active upstream sequences upstream of well repressed promoters may increase their strength to a level comparable with phage promoters.Upstream elements The flanking DNA regions of core promoters play an important role in determining transcription efficiency. but without the drawbacks associated with phage polymerase expression. The major enzymes involved in mRNA degradation are two 3’-5’ exonucleases . mRNA stability The prokaryotic mRNAs are unstable with a typical halflives ranging between 30 s to 20 min. 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.

Fusion of the ompA 5’ UTR to a variety of heterologous mRNAs significantly increased transcript half-life. Shine-Dalgarno sequence Initiation 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’.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. which is most commonly AUG. followed by an initiation codon. The stabilizing effect conferred by untranslated 5’ hairpins was first demonstrated in the case of the long-lived ompA mRNA. presumably by interfering with RNase E binding.

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

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

Coli as an Expression host E. Although E. coli has been successfully used for the expression of recombinant proteins because of its well characterized genetics and growth conditions. coli is a widely used expression system it has some disadvantages. By using well-established cultivation strategies of high cell density cultivation. 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. . a number of proteins have been produced at gram levels . E.E.

The proteins produced in E. immunogeneticity and receptor binding . coli derived products is a major problem and its consequent removal is mandatory as they are ubiquitous pathogenic molecules. coli are not glycosylated. The contamination of endotoxins in E. solubility.Sometimes. serum half-life. Glycosylation even when it is not necessary for biological activity. 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. thermal stability. often increases the stability of proteins and influences reaction kinetics.

Poor refolding yields High cost of solubilization. Beneficial for toxic protein expression. Refolding required for active protein. No need for cell lysis Dilution of the product Possible to obtain authentic N-terminus Proteolysis of recombinant protein avoided. Cytoplasmic expression Inclusion bodies are easy to (Inclusion bodies) purify. possible Usually active protein High level of intracellular product can be harmful to host cells Complex and costly purification Protein proteolysis might occur. Protection from degradation by proteases. Periplasmic expression Disulfide bond formation possible Reduced level of contaminants Possible to obtain authentic N-terminus Secretion to periplasm not always possible Periplasmic protease can cause proteolysis No large-scale procedure possible for selective release of heterologous protein from periplasm. Extracellular production Disulfide bond formation Secretion to the medium usually not possible possible. High production yields are usually obtained. Cytoplasmic expression No need of solubilization Disulfide bond formation usually not (Soluble expression) and refolding.Expression strategy Advantages Disadvantages Normally no authentic N-terminus. .

Production mode •Batch •Fed batch •Continuous cultures .

The problems arising due to high cell density are accumulation of by-product. The presence of excess glucose and oxygen limiting conditions during . 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. limitation of dissolved oxygen concentration. Problem of acetate production in E. under the demanding conditions of HCDC. Unfortunately. 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. poor mixing and degradation of product.Factors affecting high cell density cultivation Though HCDC fed-batch cultivation is a popular cultivation strategy. it is associated with certain limiting factors. increase of temperature.

5 g/l of acetate reduces growth rate.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. biomass yields and maximum attainable cell densities in high cell density cultures (as well as specific product formation rate . Accumulation of > 0.

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

However pure oxygen is expensive and higher concentration of oxygen is toxic to cells. Increasing the aeration rate or agitation speed can increase the dissolved oxygen concentration. These strategies can be combined to achieve a high cell density of various E.b) Dissolved oxygen limitation The dissolved oxygen concentration becomes limiting in high cell density cultivation owing to its low solubility. In HCDC the supply of pure oxygen has also been used. As oxygen consumption increases with the growth rate. Oxygen starvation has critical effects on cell health that some times leads to cell lysis and protease activation. the oxygen demand of cells can be reduced by lowering the growth rate. coli cultures .

c). Cultivation Temperature Temperature is an important variable that can be used to control cell metabolism. which enables higher cell-densities to be obtained without the need of pure oxygen. nutrient uptake and growth rate can be reduced. 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. Lowering of cultivation temperature also reduces cellular oxygen demand. By lowering the culture temperature from 37 oC to 26-30 oC. thus reducing the formation of toxic by-products and the generation of metabolic heat. .

The reduced growth rate also helps in obtaining higher product formation rates. a). There are two feeding strategies for the control of the nutrient feed.Robust feeding optimization strategies and process Fed-batch cultivation is usually done in two phases. . 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). Open loop control and the closed loop feed back control. a). The reduced growth rate is necessary to prevent the accumulation of inhibitory by-products. Substrate limited fed-batch strategies are common for high cell density cultivation.

Feeding strategies like feeding at constant. but also the cell productivity . The feeding strategy not only affects the maximal achievable cell density. an exponentially increasing feeding rate has to be applied in order to maintain a more or less constant specific growth rate. 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. After a batch phase without feeding.Open loop feeding Open-loop fed-batch strategies use a certain predetermined feeding profile. In an exponential feeding strategy a concentrated feed is fed at an exponential rate to maintain a predetermined .

.Closed loop (Feedback) feeding For closed-loop feedback control of physicochemical and environmental parameters. pH Stat and Carbon evolution rate (CER) are common closed loop feeding strategies having wide application in high cell density cultivation. well-known closed-loop controllers (PID-proportional-integraldifferential controller. pH. pressure. Hence a feeding strategy to maintain a constant dissolved oxygen concentration by feeding concentrated feed is used for . foam. switching controllers and others) are well established in bioprocess optimization. The use of DO Stat. Critically low DO concentrations can effect the recombinant protein production in fed-batch cultures. DO stat feeding Dissolved oxygen (DO) is an important parameter to measure the cellular health. such as temperature. stirrer speed etc.

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.This strategy is based on the finding that the DO value sharply increases when the growth limiting substrate is consumed. When complex carbon and nitrogen substrate such as yeast extract. 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. peptone and tryptone are used together with the carbohydrate substrate.

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

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