Protein expression (biotechnology)

As overexpression is called the strongly increased expression of a gene in a cell. This results in a correspondingly increased synthesis of the protein encoded by this gene.

A natural overexpression can occur in the context of a viral infection caused by a faulty gene regulation in tumors or artificially induced in the laboratory by means of molecular biological techniques to produce a targeted recombinant protein.

• 4.1 vectors
• 4.2 induction
• 4.3 promoters
• 4.4 Genetic conditions
• 4.5 substrate induction by IPTG
• 4.6 Suitable promoters for the overexpression

Applications targeted overexpression

There are two areas of application of an over-expression for the production of a recombinant protein:

• Overexpression of the protein for subsequent purification and re-use (you can still distinguish between economic processes and processes that generate small amounts of protein for research purposes should)
• Overexpression of analysis of the protein and its properties and the effect on the production organism

In analytical overexpression less emphasis is placed on economic efficiency of the process. No optimization of production, but it is referenced in the rule on proven standard, commercially available systems. An Upscale, ie high levels of quantity is not provided.

With the establishment of production processes often carried out mostly optimizing overexpression in order to obtain as stable and productive process. These varying various parameters on the genetic process and the technical level to obtain the largest possible amount of product at the end.

Prokaryotes

The most common form of the protein over-expression in prokaryotes, the use of plasmid vectors. Here, an expression cassette is installed on a plasmid (localization), transformed into the body and stabilizes there over the cultivation period. The advantage of this method lies in the genetically simple operation and high gene dosage which is achieved via the plasmid copy number.

Another method of over-expression in prokaryotes is the genomic incorporation of overexpression cassettes. For overexpression in this case must be either a strong promoter system, a high gene dosage can be achieved by multiple integration of the cassette into the genome possibly combined with RNA stabilizing modifications, elected or.

Also possible is the localization of genes for overexpression in phage genomes. With the genetically modified phage expression system is then infected. As part of the acquisition of the metabolism of the bacterium by the phage and the target gene expression is increased.

Eukaryotes

In eukaryotes, genes are overexpressed. This can be done either in cell culture or in whole organisms. For overexpression in the entire organism or transient genome- integrated expression cassettes are used. When expressed in cell culture and plasmids used. Here, large amounts produced in bacterial plasmid DNA, containing the corresponding expression cassette is introduced into the cell. The plasmids may, however, in the eukaryotic cells do not replicate and therefore will be lost after some time. This method is often used for research purposes in order to characterize the effect of over-expression in a cell. Production strains are usually created through genomic integration of the cassette.

Cell-free expression

This new form of the expression is based on the preparation of cell lysates were mixed with plasmids, and then cell-free produce the encoded on the plasmid target gene (in vitro). This method is still relatively inefficient. It can be used to produce highly toxic products.

Various types of expression

When overexpression, there are basically two ways of expression:

• Continuous expression
• Induced expression

In the continuous expression of the gene on a constitutive promoter is expressed throughout the corresponding protein and accumulates within the cell.

In the induced expression of an inducible promoter is used. By an inducer, the expression of the target gene is induced, that is, unlocked. This method is often used when the overexpression has a negative impact on the production organism. You may be a high burden of metabolic resources during the growth phase. The result is slower growth and longer terms of the bioreactor and thus specifically associated with industrial processes, an increase in production costs. Another advantage is, an induced expression in cell- toxic products. Here, it is after induction of the expression of a self- poisoning and death of the cell. With regard to the economics of the production process is therefore attempted to divide the process into a growth phase and a production phase. In the growth phase as large as possible amount of biomass is produced, and in the production phase, the target protein is then produced by induction of the promoter. In this way a maximum amount of product obtained before the death of the cells, which makes a lot more efficient the process.

Common systems for overexpression

The most frequently used system for the overexpression of recombinant proteins is the Gram-negative bacterium E. coli. But there are now a variety of other organisms used as the Gram-positive Bacillus megaterium, Corynebacteria, and yeast. Various proteins for expression in eukaryotes, it is necessary and therefore, various culture cell lines such as animal cell lines (such as Sf9 cells, CHO cells, Vero cells or HEK cells ) are used. Some proteins are produced in the course of Pharming also in genetically modified organisms, such as plants. More rarely, whole organisms of higher eukaryotes such as goats or cattle are used.

Targeted overexpression by substrate induction in E. coli

The selective expression of proteins in cells of model organisms (e.g., E. coli) with the aid of transgenes is a possibility of biological and medical research and a large amount (several mg) to produce a protein in order to examine its function or structure in further experiments. A common method is the overexpression of a transgenic, whose transcription is regulated by the own bacterial lac operon.

Vectors

The most commonly used vectors are based on pBR322 plasmid vector and adjusted by design, such as the pUC - plasmids, or pET vectors.

Induction

The most widely used system for inducing promoters is the lac operon of E. coli. This either lactose or IPTG is used as an inducer. But even systems with arabinose (see pBAD system) or rhamnose ( see E. coli KRX ) as an inducer are common. A system for physical induction, for example, the temperature -induced cold shock promoter system based on the E. coli promoter of cspA Takara.

Promoters

The selection of commercial systems with different promoters is relatively large. There are both natural promoters used, such as the T7 promoter or the bla promoter used, but also synthetic promoters and hybrid promoters such as the tac promoter, a hybrid of the promoters trp and lac from the genome of E. coli.

Genetic conditions

The transgene is located outside of the bacterial genome in a plasmid. Before the actual coding region begins, ie upstream of the transgenic, the operator is the lac operon ( lacO ). The plasmid also contains the lac gene coding for the lac repressor LacI. LacI binds to the operator lacO before the transgene and the promoter blocked, so preventing the expression of the transgene, and hence the over-production of the encoded protein.

Substrate induction by IPTG

IPTG, the allolactose a very similar molecule, binds the repressor, the operator sits on the front of the transgene. This changes the conformation of the repressor, it can no longer bind DNA and gives the operator releases. This makes available the promoter for the RNA polymerase. Binding to the promoter initiates the transcription of DNA into mRNA.

Suitable promoters for the overexpression

The release of a promoter for the initiation of transcription is not sufficient to produce very large quantities of a protein. For this purpose, large amounts mRNA to be present, ie, the transcription must run very efficiently. It takes strong promoters in rapid succession recruit RNA polymerase to the promoter and allow for both quick start of transcription. Examples include the T7 promoter from the same bacteriophages or the tac promoter, a hybrid of the trp and lac promoters from the genome of E. coli. For the T7 promoter, however, the corresponding T7 RNA - polymerase in the genome of the expression system must be present.