A group of scientists from the MIPT Laboratory of Advanced Studies of membrane proteins proposed method, which will significantly simplify the preparation of valuable proteins for further study. The approach outlined in the PLOS ONE journal can significantly reduce the cost and duration of the research, which sometimes last for many months.
The conducted study relates to the field of structural biology, one of the key objectives of which is to obtain structures of various proteins. There are from 20 to 25 thousand of them encoded only in the human genome according to various estimates. The most interesting thing for scientists and pharmacists are the proteins through which cells "communicate" with the outside world the membrane proteins that make up about a quarter of the total number of encoded proteins. However, the structure of only 3% of membrane proteins has been known for now, while scientists have found the structure of about a half of human proteins. But these are the membrane proteins in most cases, which are the targets for drugs, and a lot of money, a lot of efforts of scientists and pharmaceutical companies worldwide are spent to study them. US scientists Robert Lefkowitz and Brian Kobilka won the Nobel Prize in 2012 for obtaining the structure of specific membrane proteins the receptors conjugated with G-proteins.
It is necessary to obtain a protein in sufficient quantities first to decode its structure. The simplest and cheapest method for this is the expression in cells of E. coli the Escherichia coli, unpretentious and most studied bacteria. A gene encoding a desired protein is introduced into cells of E. coli for that, making the bacteria hyperexpress the protein that is synthesize in large quantities. The protein is isolated from the bacteria then, purified and crystallized to restore the structure of the protein with the help of the image of X-ray scattering.
Serious problems can arise already in the first stage the expression, when it is necessary to obtain the structure of the target protein. They are solved by trying different methods known at the moment, which is quite long and expensive. The authors of the article have proposed a clear algorithm that will allow solving the problems of expression with a systematic method, which greatly boosts this stage of research.
Alike (homologous) protein which expression goes better (this protein is called a driver of the expression, or a driver) is chosen for a protein, which have problems with expression. Then the chimeras, which are "stitched" from the parts of the target protein and the driver so that it is possible to determine rather quickly which site of the target protein is "guilty" of a low level of expression, are synthesized.
"It is possible to make two different chimeras replacing one half of the target protein with a half of driver. Then we check an expression of the chimeras we obtained. Based on that, which of them is best expressed, we determine in which part of the protein a place that prevents expression is. Then go to the second iteration, making two new chimeras on the basis of that chimera, which is better expressed in the first iteration, and thus twice reducing the part of driver in this chimera. We are testing the expression of new chimeras until we find out what site blocks the expression ... And so on, until we find out what exactly the problem is", Dmitry Bratanov, the first author of the article published in the PLOS ONE journal, says.
As a result, the mutation necessary for 2lg2N protein expression is found, while its random search requires 2N iterations (N is a number of amino acids in its chain).
The advantage of the new algorithm is illustrated by a small protein of 200 amino acids: it is required to synthesize not more than 16 different chimeras for it, whereas in case of iterating it is necessary to synthesize about 1060 different proteins more than in all living organisms on the planet.
Using this algorithm, the researchers from MIPT have obtained chimera of bacteriorhodopsin from the H. halobium bacterium. Its structure has been known for a long time, but at the same time it exudes from its native cells, work with which is quite difficult and requires more time than working with E. coli does. Scientists have been trying to express bacteriorhodopsin in E. coli for about 30 years, but until now the methods used for this task have not allowed to obtain it in large quantities and in a form in which the protein functions in the cell.
Bacteriorhodopsin is an important model protein to test the diverse theories associated with membrane proteins in general. The algorithm proposed by scientists will allow receiving it without using exotic techniques of expression that will significantly simplify the access to work with membrane proteins in laboratories all over the world. In addition, there are several tens of inventions based on bacteriorhodopsin used in various fields from biomedicine and biotechnology to creating of optical devices (lasers, for example) and measuring systems.
Obtaining of proteins the sequence of which is somehow different from the original one, is a standard method for improving of expression, but until now this method has been modified individually for each protein.
"Basically, the strategy of previous approaches is following. Different polypeptide sequences (tags), which can be expressive, crystallizing ones etc., are additionally placed at one end of the protein. Herewith a "hit or miss" strategy is used. If you have a good luck then a protein became expressed, if you have no luck try the following tag. In most cases, such a sequence of protein is removed during the cleaning process. We proposed an approach that allows a systematic way to identify problems that lead to a lack of protein expression. It is expected that the obtained chimeric protein will have minor changes compared to the target protein", Valentin Gordeliy, the main author of the study, says.
Using of the proposed method will significantly accelerate the process of investigation of membrane proteins in the long term that can change the strategy for the synthesis of drugs and will enable the discovering of new active ingredients by computer modelling that act faster and more accurately.
The study of membrane proteins is important for optogenetics, the new science, which already now opens up new possibilities for the study of neurodegenerative diseases Alzheimer or Parkinson ones. Results of the study will be in demand by the Center for Aging being created at MIPT.STRF.ru