Expression vector
Low level of constitutive protein synthesis may occur even in expression vectors with tightly controlled promoters.
Such insoluble proteins will require refolding, which can be an involved process and may not necessarily produce high yield.
[6] Proteins which have disulphide bonds are often not able to fold correctly due to the reducing environment in the cytoplasm which prevents such bond formation, and a possible solution is to target the protein to the periplasmic space by the use of an N-terminal signal sequence.
[12] Note that most commonly used lac or lac-derived promoters are based on the lacUV5 mutant which is insensitive to catabolite repression.
[19] Another approach would be to use a single two-cistron vector or design the coding sequences in tandem as a bi- or poly-cistronic construct.
[23] The plasmids may contain elements for insertion of foreign DNA into the yeast genome and signal sequence for the secretion of expressed protein.
Another yeast used for protein production is Kluyveromyces lactis and the gene is expressed, driven by a variant of the strong lactase LAC4 promoter.
Baculovirus, a rod-shaped virus which infects insect cells, is used as the expression vector in this system.
[26] Insect cell lines derived from Lepidopterans (moths and butterflies), such as Spodoptera frugiperda, are used as host.
[27][28] The shuttle vector is called bacmid, and gene expression is under the control of a strong promoter pPolh.
In general, it is safer to use than mammalian virus as it has a limited host range and does not infect vertebrates without modifications.
Many plant expression vectors are based on the Ti plasmid of Agrobacterium tumefaciens.
It is of particular use in producing membrane-associating proteins that require chaperones for proper folding and stability as well as containing numerous post-translational modifications.
The downside, however, is the low yield of product in comparison to prokaryotic vectors as well as the costly nature of the techniques involved.
Its complicated technology, and potential contamination with animal viruses of mammalian cell expression have also placed a constraint on its use in large-scale industrial production.
[40] E. coli cell lysate containing the cellular components required for transcription and translation are used in this in vitro method of protein production.
[43] Biotechnology allows these peptide and protein pharmaceuticals, some of which were previously rare or difficult to obtain, to be produced in large quantity.
Examples from the past include prion contamination in growth hormone extracted from pituitary glands harvested from human cadavers, which caused Creutzfeldt–Jakob disease in patients receiving treatment for dwarfism,[44] and viral contaminants in clotting factor VIII isolated from human blood that resulted in the transmission of viral diseases such as hepatitis and AIDS.
[45][46] Such risk is reduced or removed completely when the proteins are produced in non-human host cells.
Expression vectors have been used to introduce a vitamin A precursor, beta-carotene, into rice plants.
This process has also been used to introduce a gene into plants that produces an insecticide, called Bacillus thuringiensis toxin or Bt toxin which reduces the need for farmers to apply insecticides since it is produced by the modified organism.
The treatment is still a risky option due to the viral vector used which can cause ill-effects, for example giving rise to insertional mutation that can result in cancer.
