What is the functional protein of cells

One of the greatest challenges in the field of genetics is to evaluate genetic variants with regard to their functional effect on the translated protein. The entire genome of every human being contains many thousands of '' mutations '', which ultimately make up the genetic diversity of the different individuals. The vast majority of these '' mutations '' are by no means to be regarded as pathogenic, they ensure that every person is genetically unique. These non-pathogenic '' mutations '' are also known as genetic variants and occur in a larger percentage of the total world population.

The difficulty now lies in evaluating variants that only occur in a very small part of the population and therefore may be pathogenic, that is, disease-causing. Most of these variants are so-called missense variants, in which one nucleotide is exchanged for another in the DNA. This can lead to an exchange of the corresponding amino acid in the resulting protein. Whether this has an effect on the correct functioning of the protein and is therefore potentially pathogenic depends, for example, on how similar the exchanged amino acids are to one another. These things can be simulated on the computer to a limited extent, but the reliability of these predictions is still very limited.

The currently most reliable way of analyzing the effect of an amino acid exchange is therefore to simulate and examine the respective change in vitro. For this purpose, the corresponding missense variant can be simulated using a so-called mutagenesis PCR, integrated into a vector and expressed heterologously in human cell lines. A large number of experiments can then be carried out with these transfected cells: A common example of this is the luciferase assay, in which the ability of a transcription factor to bind to its target DNA is checked. Furthermore, Western blots can be carried out to check the intensity of protein expression or to co-express interacting proteins. An additional option for functional analyzes is immunohistochemical or fluorescence staining, which can be used to determine the location of expression of certain proteins in tissues or cells.