With the development of genetic engineering, a large number of bacterial organisms , fungi , insect or mammalian cells were used for the production of foreign proteins, in particular pharmaceutical proteins such as insulin or vaccines . Preferred systems for such a production are the intestinal bacterium Escherichia coli , various yeast species or cell lines of insect or mammalian cells.
A system used for the genetic production of proteins should meet various requirements: It should be able to grow quickly in large fermenters as cheaply as possible. It should efficiently produce the desired substances with the correct post-translational modifications and, if possible, discharge them into the culture medium ( secrete ). It should meet high safety requirements, especially for the production of pharmaceuticals, and produce the proteins in an authentic, preferably “human” form.
Most often, the genetic information for the protein is cloned into a vector , e.g. B. a plasmid , which is then transformed or transfected into the host organism . The properties of the recombinant protein can be adapted through protein engineering . The vector design allows for customization of the vector.
After a transgene has been cloned ( insert ) into a vector, the recombinant DNA produced is introduced into an organism. Occasionally, a clone is isolated by limiting dilution cloning as a genetically identical starting point for a cell culture . Reporter genes are occasionally used to facilitate identification of a transgenic clone . The subsequent overexpression of the recombinant protein allows higher yields than those found in the original organism. Selection markers in the vector allow the selective growth of only the transgenic organisms, while organisms that do not carry the vector with the transgene cannot grow.
For proteins, which for their expression organism are toxic, are inducible promoters used, to grow up the cells to the induction of gene expression allow. After a growth phase of the toxic genes are induced, "harvested" the cells and the addition of protease inhibitors digested . In the course of protein purification , the desired protein is separated from cellular proteins and other biomolecules and then characterized . Often proteolysable protein tags are used for cleaning and detection , which are then separated.
- Friedrich Lottspeich , Joachim W. Engels (Ed.): Bioanalytik . 2nd edition, Spektrum Akademischer Verlag, Heidelberg 2006, ISBN 978-3827415202 .
- Hubert Rehm , Thomas Letzel: The Experimenter: Protein Biochemistry / Proteomics . 6th edition, Spektrum Akademischer Verlag, Heidelberg 2009. ISBN 978-3-8274-2312-2 .
- Cornel Mülhardt: The Experimenter: Molecular Biology / Genomics. Sixth edition. Spectrum Akademischer Verlag, Heidelberg 2008. ISBN 3-8274-2036-9 .