There exist a number of expression systems and the decision on which to use depends on a range of factors. Ease of use is one important consideration as well as cost. Generally, Escherichia coli and yeast systems are explored at first, followed by insect and mammalian systems. Cultures are grown in vessels ranging from cell culture dishes, to shaker flasks and bioreactors.

Bacteria

Shortly after the discovery of the lac operon, non-metabolisable analogues of lactose such as isopropyl-β- D -thiogalactoside ( IPTG), that relieve repression of the lac repressor, were exploited to drive transcription and translation of the desired gene in Escherichia coli . The main advantage is the relative ease to manipulate, transform and induce bacteria to produce a protein of interest.

Bacteria have cell diameters of the order of 1 to 4 μm and generally have extremely rigid cell walls. Bacteria can be classified as either Gram-positive or Gram-negative, depending on whether or not they are stained by the Gram stain ( crystal violet and iodine). In Gram-positive bacteria ( Figure1 ), the plasma membrane is surrounded by a thick shell of peptidoglycan (20–50 nm), which stains with the Gram stain. In Gram negative bacteria (e.g. Escherichia coli ), the plasma membrane is surrounded by a thin (2–3 nm) layer of peptidoglycan, but this is compensated for by the presence of a second outer membrane of lipopolysaccharide. The negatively charged lipopolysaccharide polymers interact laterally, being linked by divalent cations such as magnesium. Gram negative bacteria can secrete proteins into the periplasmic space as well as the cytoplasm.

Fig1. The structure of the cell wall of Gram-negative and Gram-positive bacteria. LPS, lipopolysaccharide.

Transformation and Culturing of Bacteria

When the plasmid containing the gene of interest has been cloned into the appropriate expression vector, it is introduced (transformed) into the bacterial cells. This can be performed by applying a heat shock or a short pulse of electricity (electroporation). In either method, the brief stress to the bacteria momentarily renders the membrane permeable and allows the plasmid entry into the cell. To improve cell viability, the initial culturing conditions are gentle, by use of enriched culture media. This procedure is conducted using a small amount of medium before plating out onto an Agar plate. The plasmid will typically contain an antibiotic resistance cassette that allows selection using an appropriate antibiotic infused into the agar plate. The purpose of this method is to select a clone that has one identical copy of the plasmid that can then be expanded to a larger volume of medium, suitable for induction and to yield a substantial amount of protein.

Optimising Expression

The successful over-expression of recombinant genes in bacteria is not guaranteed. The lack of success is often due to the sequence of amino acids containing significant secondary structure that inhibit correct protein folding . Also, the gene of interest may utilise rare codons that exist in the source organism, but do not occur in the bacterial expression organism. Bioinformatics analysis coupled with high-throughput DNA synthesis techniques can be used to optimise the codon usage of the gene of interest to use the codons available in E. coli, thereby increasing the chances of successful expression.

A successful protein purification is more likely to be achieved if the starting mate rial contains a high percentage of the over-expressed protein in relation to the total protein mixture. It is therefore prudent to design trial induction experiments to ascertain the optimal expression conditions. Small-scale trials would cover a range of different temperatures, different strains of bacteria and concentrations of induction agent (for example, IPTG).

The media used for growing the bacterial culture contains the appropriate antibiotic for selection and the cells are typically grown in a shaker flask until an optical density of OD₆₀₀ = 0.5–1.0 is achieved, in order to avoid cellular stress that may be caused by over-expression of the target gene. However, T7 RNA polymerase (required for the most commonly used T7 promoter system in biotechnologically used plasmids) is expressed at a basal level in most strains of E. coli and even small amounts of expressed protein may induce cellular stress. Therefore, the choice of bacterial strain has a large impact on protein expression. Several commercially available optimised bacterial strains exist that contain plasmids encoding rare codons, strong repressor function to avoid basal expression and are highly competent (i.e. easy to transform). Also, oxygenation of the culture media during growth increases the yield, hence cultures are typically grown in shaking flasks. Flasks that include baffles will agitate the medium more to increase oxygenation.

One may encounter robust expression of a protein, when assessing entire bacterial cells by SDS-PAGE, but the protein could have been directed towards inclusion bodies, rendering the protein insoluble. This solubility problem can be addressed by cotransformation of helper plasmids that prevent the over-expressed protein becoming incorporated into inclusion bodies; examples include plasmids coding for GroEL and GroES. These molecular chaperones aid solubility of proteins in the cytoplasm by assisting in protein folding.

Media

 Luria–Bertani (LB) also known as Lysogeny broth is the most commonly used medium to culture bacteria for protein over-expression. It contains a broad range of nutrients that are derived from the enzymatic digestion of casein with trypsin (known as tryptone), yeast extract and sodium chloride in a 2:1:1 ratio (by weight). Before one can grow large cultures, the transformed bacteria are grown in an enriched medium called SOC that allows viable bacteria to grow before plating out. SOC is similar to LB, but has different ratios of nutrients and is supplemented with KCl, MgCl₂ , MgSO₄ and glucose.

There is a deliberate absence of activating sugars in LB in order to prevent basal expression of the plasmid, so that IPTG can be added to cause protein expression. However, auto-induction media can be employed that contain a carefully defined amount of glucose, that is preferentially used by the bacteria until becoming exhausted at the point just before saturation. At this moment, other sugars will induce their operons and cause expression of the target gene without the need to add IPTG.

Yeast and Fungi

Filamentous fungi and yeasts have a rigid cell wall that is composed mainly of poly saccharide (80–90%). In lower fungi and yeast, the polysaccharides are mannan and glucan; in filamentous fungi, it is chitin cross-linked with glucans. Yeasts also have a small percentage of glycoprotein in the cell wall, and there is a periplasmic space between the cell wall and the cell membrane. If the cell wall is removed, the cell con tent, surrounded by a membrane, is referred to as a spheroplast.

Mammalian Cells

Mammalian cells are of the order of 10 μm in diameter and are enclosed by a plasma membrane, weakly supported by a cytoskeleton. These cells therefore lack any substantial rigidity and are easy to disrupt by shear forces. The major advantage of using mammalian cells is the increased chances of achieving correct folding and the post-translational modifications that may be required. The disadvantages are the high costs of media and the potentially more complex chromatography protocols required.

Plant Cells

Plant cells are of the order of 100 μm in diameter and have a fairly rigid cell wall, comprising carbohydrate complexes and lignin or wax, that surrounds the plasma membrane. Although the plasma membrane is protected by this outer layer, the large size of the cell still makes it susceptible to shear forces.

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