Expression vector

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An expression vector is a nucleic acid that is used to produce recombinant proteins through overexpression . Expression vectors belong to the vectors . In contrast to expression vectors, cloning vectors are not suitable for expressing proteins, but only for cloning .

properties

Expression vectors have an operon so that an mRNA with a protein-coding sequence is generated in a suitable host as an expression system or by cell-free gene expression . A protein is generated from the mRNA in the course of translation . In addition, expression vectors have an origin of replication for the replication of the DNA, a sequence for the selection of the expression vector (e.g. antibiotic resistance , inactivation of a toxic gene or auxotrophy ) and a polylinker into which the protein-coding sequence of the desired protein (the insert ) is inserted. If the protein to be expressed is itself toxic to the expression system, inducible promoters are used which allow the host to grow up to induction. Most often the DNA is used in the form of a plasmid . Often a coding sequence for a protein tag is added to the protein coding sequence, creating a fusion protein that is easier to purify and detect . Expression vectors are often additionally modified by methods of vector design .

Several proteins can also be generated in parallel in a host, provided that plasmids with different selection mechanisms are used, since only one type is necessary with the same selection mechanism and often only one type is retained by the host. Vectors with two promoters are also used. For bacteria, constructs with two cistrons can also be used. In eukaryotes, an IRES can be used to generate two different proteins in parallel between two protein-coding sequences, the yield of the protein with the protein-coding sequence usually being lower after the IRES.

Bacterial Expression Vectors

pGex plasmid for bacteria

The cheapest form of protein expression takes place in single cells . However, not all eukaryotic proteins can be produced by bacteria in their native state , as a result of which the biological activity is reduced or undesired inclusion bodies can arise, which have a negative effect on the yield in protein purification. Most Escherichia coli , Bacillus subtilis or, more rarely, Ralstonia eutropha are used as hosts . In addition to plasmids, bacterial artificial chromosomes are also used.

Bacterial expression vectors often have a promoter consisting of the lac operon (then usually the lac UV5 - mutant without catabolite ) or from the bacteriophage T7 (eg the. PET derived vector). Plasmids of the pQE type use a T5 promoter with a lower basal expression , which is better suited for host-toxic proteins. Furthermore, parts of the arabinose- sensitive promoter araBAD or of the rhamnose- sensitive promoter rhaBAD are used to control the induction of gene expression . Promoters composed of different origins are also used, such as the Tac promoter from the trp and the lac promoter, for example in the case of the pGex vector. The origin of replication of plasmids is usually those with a high number of plasmid copies . A high number of plasmid copies leads to high gene expression, but also to higher toxicity of toxic proteins.

If the protein to be expressed has disulfide bridges , a signal sequence for secretion into the periplasm is required because the cytosol has a reducing effect and prevents the formation of disulfide bridges. In most cases, a protease interface is inserted between the signal sequence and the desired protein, whereby the signal sequence is cleaved after or during the purification of the protein in order to obtain a protein that is as native as possible .

Eukaryotic Expression Vectors

Ti plasmid for plants

The cheapest expression in eukaryotes is carried out in yeasts such as Saccharomyces cerevisiae or Komagataella phaffii (formerly known as Pichia pastoris , as a vector e.g. pIC ), which can glycosylate the proteins. Unless human-like glycosylation patterns are required, one is overexpressed in insect cell culture with baculovirus vectors, or in mammalian cell cultures performed. The vectors used are plasmids, yeast artificial chromosomes (in yeasts), mammalian artificial chromosomes (in mammalian cells) or viral vectors . Although plasmids for use in eukaryotes have a eukaryotic operon, they have a bacterial origin of replication for inexpensive reproduction in bacteria. Due to the lack of eukaryotic origins of replication, plasmids are not replicated in eukaryotes, as a result of which the duration of the expression of the protein is limited because of the degradation of the plasmids ( transient expression ).

In mammalian cells, a CMV immediate / early 1 promoter or an SV40 promoter is often used, more rarely an EF-1 promoter or the chicken β- actin promoter and hybrids of several promoters. Subsequent to the protein-coding sequence, a poly-A sequence (e.g. from the gene for the bovine somatotropin bGH or from the SV40 virus) is used, which slows down the breakdown of the mRNA.

The Ti plasmid is often used in plants, or a viral vector based on the tobacco mosaic virus (TMV), the potato virus X or the cowpea mosaic virus .

use

Expression vectors are used for the production of recombinant proteins, in biochemical research or in the biotechnological-industrial production of proteins. They are also used to generate transgenic organisms on a permanent basis , for gene therapy in humans .

literature

Individual evidence

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