Research Projects All Projects
Projects

Quick links

Development of orphan G-protein coupled receptor peptidic ligand identification system

 

 

Project Title: „Development of orphan G-protein coupled receptor peptidic ligand identification system” 

Funding: European Regional Development Fund (ERDF), Measure 1.1.1.1 “Support for applied research”

Project Nr.: 1.1.1.1/16/A/055

Period: 1st January 2017 – 31st December 2019

Project costs: 285 245,84 EUR

Principle Investigator: Dr. biol. D. Fridmanis

 

The aim of this Project is to develop humanized yeast S.cerevisiae based GPCR peptide ligand identification system and to evaluate its effectiveness by de-orphanization of C.intestinalis orexin-like receptor and determination of novel ligand’s pharmacological properties. The results of this Project shall provide instrument for the development of novel pharmaceutical compounds and promote development of biopharmaceutical industry in Latvia.

Information published: 02.01.2017.

 

Progress of the project

1 January 2017 - 31 March 2017

Experimental development

According to plan the first three months of the project were devoted to creation of double expression plasmid. Since p426GPD and p426TEF expression plasmids that have been successfully employed by our group are nearly identical, then within the scope of these activities we altered the multi-clonal sites of the expression loci in a way that each would contain only two unique cloning/restriction sites, thus becoming mutually compatible. Following the modification of both plasmids we performed the sequencing analysis of both expression loci to ascertain the conformity of acquired sequences to planned ones and carried out the transfer of TEF containing locus from p426TEF to p426GPD plasmid. At the moment we are performing the sequencing analysis of thus acquired construct.

Information published: 31.03.2017.


Progress of the project

1 April 2017 - 30 June 2017

Experimental development

During this project phase the functional regions of TEF promoter containing plasmid that were relevant for gene expression were transferred to GDP promoter containing plasmid. Further within thus acquired double expression plasmid MC4R and a-MSH coding genes were inserted and following their insertion conformity of acquired results with the plans was established by sequencing analysis. After acquisition of expression constructs we commenced with evaluation of its functionality.

Information published: 30.06.2017.

 

Progress of the project

1 July 2017 - 30 September 2017

Experimental development

During this project phase we initiated the functional testing of previously created double expression plasmid. Within the scope of these activities the newly created plasmid was inserted into the cells of humanized yeast S.cerevisiae strain MMY28, which were subsequently seeded within the selective media containing petri dish. The media within selected dishes was supplemented with MC4R ligand - a-MSH as the means of positive control. Acquired results were ambiguous therefore experiments shall be repeated during further project stages.

Information published: 29.09.2017.

 

Progress of the project

1 October 2017 - 31 December 2017

Experimental development

During this stage of the project we continued with the evaluation of previously created double expression. During these activities plasmids were repeated transformed within yeast S.cerevisiae strain MMY28 cells, which subsequently were seeded on peri-dishes with selective media that in several cases was supplemented with a-MSH. Acquired results, unfortunately, were again ambiguous, therefore it was decided to assess the expression activity of the plasmids employing Western-Blot with a‑MSH specific antibodies. In parallel to facilitate the progress of the whole project we developed an experimental plan that involved successive yeast S.cerevisiae strain MMY28 cell transformation with two separate plasmids.

Information published: 02.01.2018.

 

Progress of the project

1 January 2018 - 31 March 2018

 Experimental development

Within the scope of Experimental development activity experiments on evaluation previously created plasmid ability to secrete a-MSH were re carried out. These experiments involved employment of Western-Blot spotting with a-MSH specific antibody. Acquired results confirmed that a-MSH is being successfully secreted, however, since the signal was rather week, it was concluded that its amount could be insufficient to effectively activate the co-expressed MC4R, which could explain ambiguity in previously acquired results. To resolve this issue, an in-depth literature analysis was carried out and as the result it was concluded that it might be necessary to insert the linker peptide between the secretion signal and secreted peptide. T the moment the creation of this construct is being carried out. In parallel with these activities, an experiment on sequential transformation of two separate (first receptor and then random peptide) plasmids into the yeast S.cerevisiae MMY28 strain cells and their subsequent the culturing in histidine lacking media, that promotes propagation of only those cells that expressed receptor is activated. Acquired results were promising, because cells cultures that contained both plasmids were growing faster that thos that contained only the receptor expression plasmid.

System validation

Since the results that were gained within the scope of “Experimental development activity” displayed that the developed experimental system could be operational, within the scope of this activity we attempted to assess it. During these experiments we created plasmid that instead of a-MSH coding sequence contained coding sequence for randomized peptide. Further this plasmid along with MC4R coding sequence containing plasmid were successively transformed into yeast S.cerevisiae MMY28 strain cells, then cultured for several days within the histidine lacking media and extracted. Further, thus acquired plasmid DNA along with randomized source plasmid were used for generation of randomized region Next Generation Sequencing libraries and sequenced using IonTorrent PGM sequencing system. Acquired results showed that selection of clones which contained a-MSH coding sequence has taken place during the randomization and not culturing stage. Therefore during the next project stages improvements in randomization process shall be made.

Information published: 29.03.2018.

 

Progress of the project

1 April 2018 – 30 June 2018

Experimental development

During this period an expression plasmid that contained linker between secretion signal and peptide. In next project periods experiments shall be carried out to evaluate its peptide secretion efficiency. In addition a new strategy was developed for replacement of a-MSH coding sequence with randomized peptide coding sequence and first experiments were initiated to evaluate the system’s efficiency.

System validation

Although the previous validation experiments displayed significant drawbacks in randomization, they resulted in acquisition of rather large data set. Therefore during this project period in-depth data analysis was performed. Acquired results showed that within data set, which was gained after yeast culturing, the proportion of sequences that coded melanocortin receptor pharmacophore was slightly higher than within dataset, which was gained from randomized plasmid, thus indicating that even in the case of suboptimal randomization the system is working and selection of specific clones in taking place.

Information published: 29.06.2018.

 

Progress of the project

1 July 2018 – 30 September 2018

Experimental development

During this project period, experiments for testing of the new randomization strategy were performed. Within their framework whole plasmid amplification, employing newly created primer pairs, followed by ligation and transformation were performed. However, in the end, only a small number of plasmid-positive colonies were obtained. However, although these results were not promising following cultivation, next generation sequencing of the randomized plasmid and subsequent data analysis was performed. As a result, it was uncoveredthat the randomization process was essentially successful, as the acquired reads didn’t contain a-MSH coding sequence. Therefore, in order to increase the number of positive colonies of plasmid, a further improvement of the randomization process, which increased the number of colonies by 10 fold, was performed. However, this result also didno’t reach the required randomization level. For this reason, further improvement attempts have been initiated, the result of which will be identified at the next stage of the project.

System validation

During this project phase system validation experiments were performed using the most successfully randomized plasmid that was created during the Experimental Development activity. Thus following a sequential transformation with MC4R and randomized plasmids yeast cells were cultured for two weeks in environment without histidine. After completion of the experiment, the selected plasmid was isolated from culture and currently its sequencing analysis is performed.

Information published: 28.09.2018.

 

Progress of the project

1 October 2018 – 31 December 2018

Experimental development

At the beginning of this project period, a method for obtaining a randomized plasmid was developed It included employment of DNA oligonucleotide with first six 5 'end nucleotides replaced to ribonucleotides. Within the framework of these works, a complete plasmid amplification reaction was performed and the resulting product was treated with RNase H, which specifically cleaves only the RNA within double-stranded RNA-DNA hybrid, thus creating a 3' overhang. After acquisition of thus created plasmid, it was transformed into E. coli cells and seeded on a petri dish. Unfortunately, the efficiency of plasmid randomization did not differ significantly from the effectiveness of the initial attempts. Therefore, an in-depth analysis of the results were carried out and it was concluded that the oligonucleotide RNA part is subject to auto degradation due to temperature changes during amplification, which prevents the formation of overhang and reduces the efficiency of the fragment end binding. Therefore, in the second part of the project period, an alternative randomization strategy was developed, which included the replacement of deoxythymidine, which is located close to 5 'end with deoxyuridine and following treatment with UNG.

System validation

During this phase of the project, an analysis of previously acquired sequencing results was carried. The obtained results showed that in essence the ligand selection system is functional, since the sequence reads that were obtained after cultivation contained the a-MSH coding sequence, while such were not found in the randomized plasmid reads.

Information published 28.12.2018.

 

Progress of the project

1 January 2019 – 31 March 2019

Experimental development

During this project period, the first experiments were carried out using randomization oligonucleotides that contained deoxyuracil at their 5 'end. However, all the amplification attempts were unsuccessful. Given that most of the previously performed experiments of whole plasmid amplification were successful, it was concluded that the employed randomization primer  has a very strong secondary structure that prevents its binding to the plasmid. This assumption was also confirmed by the in-silico simulation of its structure. Therefore, using the same principles, an alternative primer variant was developed whose in-silico simulation did not display any strong secondary structure. Further experiments using this primer will be carried out in the next stages of the project.

System validation

At this stage of the project within the scope of this activity, the process of preparing sequencing libraries was optimized. Thus the yeast plasmid expraction procedure was improved and the randomized region amplification conditions were optimized.

Information published 29.03.2019.

 

Progress of the project

1 April 2019 – 30 June 2019

Experimental development

During this period, experiments involving employment of alternative deoxyuracil containing primers were completed. However, unfortunately, even in this case, all the amplification attempts were unsuccessful. In order to explain the obtained results, the analysis of scientific literature was carried out, in which the properties of different thermostable polymerases were compared. As a result it was concluded that the activity thus far used enzyme was adversely affected by the deoxyuracil, so all previously developed primer pairs were subjected to repeated plasmid amplification experiments using a specially selected uridine tolerant polymerase. The results of these actions confirmed the correctness of the hypothesis and the products obtained by the first primer pair were treated with uracil-DNA glycosylase to create an abasic site at the position of uracil. According to literature data, when subjected to elevated temperature such site is prone to spontaneous cleavage, resulting in formation of overhangs that contribute to circularization. However, the transformation of the resulting plasmid into E.coli cells did not show the desired increase in the number of colonies, therefore it was concluded that spontaneous cleavage is not efficient enough and it is necessary to promote it by employment of enzymes. Further, in order to achieve the desired result a mixture of USER enzymes was purchased from the company “NEB” and its employment significantly increased the number of transformation positive colonies, thus successfully completing experimental development activity.

System validation

During this period preparation of sequencing libraries was significantly improved and we developed primers that shall enable the employment of BMC's recently acquired Illumina MiSeq sequencer, which is more accurate sequencing system than previously used IonTorrent PGM device. In addition, the plasmid extraction procedure from yeast cells was also significantly improved, which shall significantly improve the reproducibility of future results.

Information published 28.06.2019.



Mājas lapas izstrādi finansēja ERAF 2.1.1.2. aktivitātes projekts Nr. 2010/0196/2DP/2.1.1.2.0/10/APIA/VIAA/004 "Latvijas biomedicīnas pētījumu integrācija Eiropas zinātnes telpā".