Chimeras will simplify the synthesis of proteins
MIPT biophysicists together with their foreign colleagues
we learned how to synthesize valuable proteins quickly with the help of chimeras
A group of researchers from the Laboratory of Advanced Research of Membrane Proteins of the Moscow Institute of Physics and Technology (MIPT) and their foreign colleagues from the Research Center of the Julich (Germany) and the Institute of Structural Biology (France), proposed a method that will significantly simplify the production of valuable proteins for further study. Described in the pages of the journal PLOS ONE (Bratanov et al., An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin) approach can significantly reduce the cost and, most importantly, the duration of research, which can now last many months, due to the systematic search for possible solutions instead of the "pan-or-gone" approach (actually random iteration of mutations), often used now.
The research done can be attributed to the field of structural biology – a field of science, one of the key tasks of which is to obtain structures of various proteins. According to various estimates, 20,000 to 25,000 of them are encrypted in the human genome alone. The proteins that are most interesting for scientists and pharmacists are the proteins with which cells "communicate" with the outside world – membrane proteins that make up about a quarter of the total number of encrypted proteins. However, at the moment, the structure of only 3% of membrane proteins is known (while the proportion of proteins with a known structure is about 50% of the total number of all human proteins). This is despite the fact that membrane proteins are in most cases the targets for drugs, and huge amounts of money and efforts are spent on their study by both scientists and pharmaceutical companies around the world. In 2012, the Nobel Prize was awarded for the study of a family of membrane proteins – receptors conjugated with G-proteins.
The structure of the complex of three bacteriorhodopsin obtained by the authors.
Three-dimensional version: http://goo.gl/l26Ljo
Screenshot: rscb.org/pdb .
To decipher the structure of the protein, first of all it is necessary to obtain it in sufficient quantity. The simplest and cheapest method for this is the expression in the cells of E.coli – E. coli, an unpretentious and most studied bacterium. To do this, the gene encoding the desired protein is injected into E.coli cells, forcing bacteria to overexpress this protein (that is, synthesize in large quantities). Then the protein is isolated from bacteria, purified and crystallized, so that later, using X-ray scattering, the protein structure can be restored.
Scientists can face serious problems already at the first stage – expression. Their solution often occurs by going through various methods known at the moment, which is often long and expensive. The authors of the article proposed an approach that will allow solving expression problems in a systematic way, based on a clear algorithm. This will significantly accelerate this stage in research around the world.
The essence of the solution is as follows. For a protein that has problems with expression (this is the target protein whose structure needs to be obtained), another protein similar to it (homologous) is selected, whose expression is better (this protein is called an expression driver, or simply a driver). Then chimeras are synthesized, "stitched" from parts of the target protein and the driver in such a way that it is quite quickly possible to determine which part of the target protein is to blame for the low level of expression.
"You can make two different chimeras by replacing one of the halves of the target protein with half of the driver. The expression of the resulting chimeras is checked. Based on which of them is expressed better, we determine in which half of the protein there is a place that interferes with expression. Then we move on to the second iteration, making two new chimeras based on the chimera that was better expressed in the first iteration, and thereby reducing the driver's share in this chimera by half. The expression of new chimeras is checked, we find out which part prevents expression... and so on, until we find out exactly what the problem is," explains Dmitry Bratanov, the first author of the article.
An illustration of the proposed algorithm. Image: PLOS ONE
It should be noted that as a result, it turns out to detect the necessary mutation in 2log 2 N protein expressions, while its random search requires 2 N iterations (here N is the number of amino acids in its chain). The advantage of the new algorithm can be seen on the example of a small protein of 200 amino acids: it will require synthesizing no more than 16 different chimeras, while a random search requires synthesizing about 1060 different proteins – more than in all living organisms on the planet.
As an example of the algorithm, the authors obtained a chimera of bacteriorhodopsin from the bacterium H.halobium. Its structure has been obtained for a long time, but at the same time it stood out from its native cells, work with which is difficult and requires more time than work with E.coli. In E.coli, scientists have been trying to express bacteriorhodopsin for about 30 years, but so far the methods that have been applied to this task have not allowed get it in large quantities and in the form in which the protein functions in the cell.
Crystals of bacteriorhodopsin – protein, on the example of which the proposed method was tested.
Image courtesy of the authors of the study
Bacteriorhodopsin itself is an important model protein for testing various theories related to membrane proteins in general. The algorithm proposed by scientists makes it possible to obtain it without using exotic expression methods, which will significantly simplify access to work with membrane proteins in laboratories around the world and greatly reduce the cost of obtaining them. In addition, there are several dozen inventions based on bacteriorhodopsin used in industries ranging from biomedicine and biotechnology to the creation of optical instruments (lasers, for example) and measuring systems.
It should be noted that obtaining proteins whose sequence differs in some way from the original one is a standard method for improving expression, but so far this method has been modified individually for each protein.
"Basically, the strategy of the previous approaches is as follows. Various polypeptide sequences (tags) are additionally placed on one of the ends of the protein, which can be expression, crystallization, etc. At the same time, the "pan-or-gone" strategy is used. Lucky – the protein began to be expressed, unlucky – let's try the next tag. Most often, in the process of protein purification, such a sequence is removed. We have proposed an approach that allows us to systematically identify problems that lead to a lack of protein expression. At the same time, it is assumed that the resulting chimeric protein will have minor changes compared to the target protein," explains Valentin Gordeliy, the author of the idea of the method.
In the future, the proposed method will significantly speed up the processes of studying membrane proteins, which may change the strategy of drug synthesis and make it possible to find new active substances faster and more accurately using computer modeling. Also, the study of membrane proteins is important for the new science of optogenetics, which already opens up incredible opportunities for the study of neurodegenerative diseases, such as Alzheimer's or Parkinson's disease.
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02.11.2015