Overexpression of prokaryotic genes

Although some useful Bacterial proteins and metabolites can be harvested from wild-type cells or their media, most are at such low levels that strains need to be improved to enhance recovery. There are four main approaches to overexpressing a particular gene or pathway:
? Mutation of other genes causing repression
? Increasing gene dosage
? Controlling gene-specific promoters
? Fusion to another protein

Frequently a combination of all four techniques is used. The cell is a complex network of repressors and activators some of which serve to repress the cellular levels of molecules or proteins that have biotechnological significance. Random or site- directed mutation can result in derepression and accumulation of bioproducts. Mutational approaches can also be used to alter cell membrane transporters, allowing the cell to use substrates not available to the wild type.

Mutational approaches can lead to significant increases in product yield at the research scale, but where the cell is being exploited as a ‘microbial factory,’ economics dictate that even higher yields are required. Cloning genes into plasmids known as expression vectors will result in more copies of the gene being present in the cell. This will lead to more mRNA and hopefully more protein. This protein can then be harvested, or may act to produce more metabolite if this is the goal. However, increasing the gene dosage in this way can result in too much protein being produced in the cytoplasm. This can aggregate to form inclusion bodies, which are hard to isolate active protein from, or the protein can block membrane transport processes. The biotechnologist thus has a range of expression vectors available, from low copy number plasmids for toxic proteins, to high copy plasmids for amenable proteins.

In addition to manipulating gene dosage, the time at which gene expression begins can be crucial. A protein expressed during early logarithmic growth may prove toxic, but can be expressed at high levels if the recombinant gene is only switched on during late log phase or stationary phase. Regulation of protein expression is achieved by using adapted promoters, placed just upstream of the gene of interest in the expression vector. The first regulated expression vectors used the lac promoter so that the gene of interest could be switched on using IPTG (isopropyl-b-D1-thiogalactopyranoside). However, better, more controllable expression could be induced by using a promoter that was a hybrid of the lac and trp promoters. This ‘trc’ promoter retains the ability to be turned on by IPTG but is of greater strength than lac alone. Other tunable promoters have been designed that turn on genes in response to low levels of uncommon carbohydrates such as xylose and arabinose. This alleviates the need to use the relatively expensive IPTG. All these different promoters allow the biotechnologist to turn on a gene merely by adding an inducer at any point during fermentation.

Even  though all these techniques can  result in  up  to  10% of a Bacterial cell’s protein being composed of the  recombinant, some medically and  industrially important proteins can be hard to purify,  cannot be expressed at high levels due  to cell toxicity,  are not stable at high  concentration, or prove to be particularly susceptible to protease activity. In these cases the recombinant protein can be ‘fused’ to another small protein to aid both stability and purification. For example, the C-terminal of a fusion protein can be made of 220 amino acids normally found in Bacterial glutathione-S-transferase (GST). After expression, this will allow the rapid purification of the fusion protein by using a column containing agarose coated with glutathione. The glutathione binds strongly to the GST domain of the fusion protein and other cellular constituents can be washed away. Although the fusion protein approach is useful in many applications, the protein of interest is physically very close to GST and this can lead to reduction or loss of activity. Inclusion of a thrombin domain between the GST and the protein of interest allows cleavage of the more valuable polypeptide away from GST. Other commonly used fusion proteins are based around the Bacterial maltose binding protein or synthetic histidine tagging genes.