Overexpression
When overexpression is defined as the greatly increased expression of a gene in a cell . This results in a correspondingly increased synthesis of the protein that is encoded by this gene .
Natural overexpression can occur in the context of a viral infection , in tumors it can be caused by faulty gene regulation or artificially induced in the laboratory to generate a recombinant protein with the help of an expression vector .
Applications of targeted overexpression
There are two areas in which overexpression can be used to produce a recombinant protein:
- Overexpression of the protein for later purification and further use (here a distinction can still be made between economic processes and processes that should generate small amounts of protein for research purposes)
- Overexpression to analyze the protein and its properties and the influence on the production organism
With analytical overexpression, less emphasis is placed on the economic efficiency of the process. There is no optimization of the production, but as a rule, tried and tested commercially available standard systems are used. An upscale, i.e. H. Increasing the amount is not provided.
When production processes are established, there is often extensive optimization of overexpression in order to obtain the most stable and productive process possible. Various parameters are varied on a genetic and process level in order to end up with the largest possible amount of product.
Prokaryotes
The most common form of protein overexpression in prokaryotes is the use of plasmid vectors . Here, an expression cassette is installed on a plasmid (localization), transformed into the organism and stabilized there over the cultivation period. The advantage of this method lies in the genetically simple way of working and the high gene dose that is achieved via the number of plasmid copies.
Another method of overexpression in prokaryotes is the genomic incorporation of overexpression cassettes. For overexpression, either a strong promoter system, possibly combined with RNA-stabilizing modifications, must be selected, or a high gene dose must be achieved through multiple integration of the cassette into the genome.
It is also possible to localize genes for overexpression in phage genomes. The expression system is then infected with the genetically modified phages. When the phage takes over the metabolism of the bacterium, the target gene is also expressed to a greater extent.
Eukaryotes
Genes are also overexpressed in eukaryotes . This can be done either in cell culture or in the entire organism. Transient or genome-integrated expression cassettes are used for overexpression in the entire organism. As promoters of be CMV promoter and CAG promoter used. Plasmids are also used for expression in cell culture. In this case, be in the cell produced in large quantities bacterial plasmid DNA, containing the corresponding expression cassette inserted . However, the plasmids cannot replicate in the eukaryotic cells and are therefore lost after a while. This method is often used for research purposes to characterize the effect of overexpression on a cell. Production strains are usually created via genomic integration of the cassette.
Cell-free expression
The cell-free protein synthesis is based on the preparation of cell lysates with plasmids or mRNA are mixed and then cell-free ( in vitro to produce the encoded target protein). This method is still relatively inefficient. It is suitable for the manufacture of highly toxic products.
Different types of expression
With overexpression there are basically two ways of expression:
- continuous expression
- induced expression
With continuous expression, the gene is continuously expressed via a constitutive promoter and the corresponding protein accumulates in the cell.
An inducible promoter is used in the induced expression. The expression of the target gene is induced, that is, activated, by an inducer. This method is popular when the overexpression has negative effects on the production organism. Reasons for this can be a high load on metabolic resources during the growth phase. The consequence is slower growth and thus longer runtimes of the bioreactor and thus an increase in production costs, especially in industrial processes. Induced expression in products that are toxic to cells is also advantageous. Here, after the induction of expression, self-poisoning and death of the cell occurs. With regard to the profitability of a production process, attempts are therefore made to divide the process into a growth phase and a production phase. The largest possible amount of biomass is generated in the growth phase and the target protein is then produced in the production phase by induction of the promoter. In this way, a maximum amount of product is obtained before the cells die, which makes the process significantly more economical.
Common overexpression systems
The most frequently used system for the overexpression of recombinant proteins is the gram-negative bacterium E. coli . However, various other organisms are now used, such as the Gram-positive Bacillus megaterium , Corynebacteria and yeasts . Expression in eukaryotes is necessary for various proteins, which is why various cell culture lines such as animal cell lines (e.g. Sf9 cells, CHO cells , Vero cells or HEK cells ) are used. Some proteins are also produced in genetically modified organisms such as plants in the course of pharming . Whole organisms of higher eukaryotes such as goats or cattle are also used less frequently .
Targeted overexpression through substrate induction in E. coli
The targeted overexpression of proteins in cells of model organisms (e.g. E. coli ) with the help of transgenes is a possibility in biological and medical research to produce a larger amount (several mg ) of a protein in order to use its function or in further experiments Examine structure . A common method is the overexpression of a transgene, the transcription of which is regulated by the bacterial lac operon .
Vectors
Most commonly, vectors based on the pBR322 plasmid are used and adapted by vector design , such as the pUC plasmids or the pET vectors.
induction
The most widely used system for inducing promoters is the E. coli lac operon . Either lactose or IPTG is used as an inducer. But systems with arabinose (see pBAD system) or rhamnose (see E. coli KRX) as an inducer are also widespread. A system for physical induction is, for example, the temperature-induced cold shock promoter system based on the E. coli cspA promoter from Takara.
Promoters
The selection of commercial systems with different promoters is relatively large. Natural promoters are used, such as the T7 promoter or the bla promoter, but also synthetic promoters and hybrid promoters such as the tac promoter, a hybrid of the trp and lac promoters from the E. coli genome .
Genetic requirements
The transgene is located outside the bacterial genome on a plasmid . Before the actual coding region begins, i.e. upstream of the transgene, the operator of the lac operon ( lacO ) is located. The plasmid also contains the gene lacI , which codes for the lac repressor LacI. LacI binds the operator lacO in front of the transgene and blocks its promoter , thus preventing the expression of the transgene and thus the overproduction of the encoded protein.
Substrate induction by IPTG
IPTG , a molecule very similar to allolactose , binds the repressor that is located on the operator in front of the transgene. This changes the conformation of the repressor, it can no longer bind the DNA and releases the operator. This makes the promoter accessible to the RNA polymerase . Their binding to the promoter initiates the transcription of the DNA into mRNA .
Suitable promoters for overexpression
The release of a promoter to initiate transcription alone is not enough to produce very large amounts of a protein. For this, large amounts of mRNA must be available, which means that the transcription must be very efficient. Strong promoters are required which recruit the RNA polymerases to the promoter in rapid succession and enable transcription to start just as quickly. Examples include the T7 promoter from the bacteriophage of the same name or the tac promoter, a hybrid of the trp and lac promoters from the E. coli genome . For the T7 promoter, however, the corresponding T7 RNA polymerase must also be present in the genome of the expression system.
literature
- Joseph Sambrook , David W. Russell: Molecular Cloning. A Laboratory Manual. Volume 3. 3rd edition. Cold Spring Harbor Laboratory Press, New York NY 2001, ISBN 0-87969-576-5 .
Individual evidence
- ↑ Marc Cruts et al. a .: Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. In: Nature. 442, pp. 920-924 (August 24, 2006) doi : 10.1038 / nature05017 .
- ↑ Ma Y, Ghoshdastider U, Wang J, Ye W, DötschV, Filipek S, Bernhard F, Wang X: Cell-free expression of human Glucosamine 6-phosphate N-acetyltransferase (HsGNA1) for inhibitor screening. . In: Protein Expres Purif . 86, No. 2, 2012, pp. 120-126. doi : 10.1016 / j.pep.2012.09.011 . PMID 23036358 .
- ^ C. Roos, L. Kai, S. Haberstock, D. Proverbio, U. Ghoshdastider, Y. Ma, S. Filipek, X. Wang, V. Dötsch, F. Bernhard: High-Level Cell-Free Production of Membrane Proteins with Nanodiscs . In: Methods in Molecular Biology . 1118, 2014, pp. 109-30. doi : 10.1007 / 978-1-62703-782-2_7 . PMID 24395412 .
- ↑ Roos, L Kai, D Proverbio, U Ghoshdastider, S Filipek, V Dötsch, F Bernhard: Co-translational association of cell-free expressed membrane proteins with supplied lipid bilayers . In: Molecular Membrane Biology . 30, No. 1, 2013, pp. 75-89. doi : 10.3109 / 09687688.2012.693212 . PMID 22716775 .
- ↑ DA Mead, E. Szczesna-Skorupa, B. Kemper: Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. In: Protein Engineering. Year 1986, No. 1, pp. 67-74.
- ↑ HA de Boer, LJ Comstock, M. Vasser: The tac promoter: a functional hybrid derived from the trp and lac promoters. In: Proceedings of the National Academy of Sciences . No. 80, 1983, pp. 21-25.
- ^ T7 promoter systems website by Sigma-Aldrich, accessed on May 29, 2011.