The GDF-11 protein was genetically modified to enhance its resistance to proteolysis and creatinine, and plasmid synthesis for its expression was carried out. Plasmid synthesis of a genetically modified protein with the conditional name GDF-11 (gene modification) was carried out at CJSC "Eurogen" (Russia, Moscow) using the method of cyclic assembly from oligonucleotides (PCA, polymerase cycling assembly). A new cross-linked protein GDF-11 (gene modification) using cyclization has been developed. Two amino acid residues of cysteine (C) based on two-component (2C) groups were introduced into the amino acid sequences of GDF-11 protein. To increase the stability of peptide sequences in the studied protein, L-amino acids were replaced with D-amino acids. To obtain the GDF-11 protein (genetically modified), the DNA matrix for the selected one must be included in a protein-specific vector, in this case, in E. coli cells. The resulting protein modification may enhance bioavailability, resistance to proteolysis and, accordingly, biological activity. Final conclusions about the effectiveness of gene modification of a known protein should be made after confirmation in vitro and in vivo experiments.

Tikhonov Sergey L., Tikhonova Natalya V., Valieva Sholpan S.

1. Pham P. V. Medical biotechnology: techniques and applications. Ch. 19. In.: Barh D., Azevedo V. (Eds.). Omics technologies and bio-engineering towards improving quality of life, Academic Press, 2018, P. 449–469. https://doi.org/10.1016/B978-0-12-804659-3.00019-1.
2. Khan S., Ullah M. W., Siddique R., Nabi G., Manan S., Yousaf M. [et al.]. Role of recombinant DNA technology to improve life. International Journal of Genomics, 2016;1:2405954. https://doi.org/10.1155/2016/2405954.
3. Klein-Marcuschamer D., Oleskowicz-Popiel P., Simmons B. A., Blanch H. W. The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnology and Bioengineering, 2012;109;4:1083–1087. https://doi.org/10.1002/bit.24370.
4. Vestergaard M., Chan S. H. J., Jensen P. R. Can microbes compete with cows for sustainable protein production – a feasibility study on high quality protein. Scientific reports, 2016;6:36421. https://doi.org/10.1038/srep36421.
5. Permyakova L., Sergeeva I., Ryabokoneva L., Atuchin V., Li Y., Markov A. Peptides of yeast Saccharomyces cerevisiae activated by the malt sprout extract: Preparation, identification and bioactivity. Food Bioscience, 2024;61:104867. https://doi.org/10.1016/j.fbio.2024.104867.
6. Sergeeva I., Permyakova L., Markov A., Ryabokoneva L., Atuchin V., Anshukov A. [et al.]. Peptides of yeast Saccharomyces cerevisiae activated by the aquatic extract of Atriplex sibirica L. ACS Food Science & Technology, 2024;4;1:173–189. https://doi.org/10.1021/acsfoodscitech.3c00455.
7. Keppler J. K., Heyse A., Scheidler E., Uttinger M. J., Fitzner L., Jandt U. [et al.]. Towards recombinantly produced milk proteins: physicochemical and emulsifying properties of engineered whey protein beta-lactoglobulin variants. Food Hydrocolloids, 2021;110:106132. https://doi.org/10.1016/j.foodhyd.2020.106132.
8. Karbalaei M., Rezaee S. A., Farsiani H. Pichia pastoris: a highly successful expression system for optimal synthesis of heterologous proteins. Journal of Cellular Physiology, 2020;235;9:5867–5881. https://doi.org/10.1002/jcp.29583.
9. Ward O. P. Production of recombinant proteins by filamentous fungi. Biotechnology Advances, 2012;30;5:1119–1139. https://doi.org/10.1016/j.biotechadv.2011.09.012.
10. Pourmir A., Johannes T. W. Directed evolution: selection of the host organism. Computational and Structural Biotechnology Journal, 2012;2;3:e201209012. https://doi.org/10.5936/csbj.201209012.
11. Gaponova L. V., Logvinova T. T., Pershikova A. V., Reshetnik E. I. Soybean in medical and preventive and children's nutrition. Molochnaya promyshlennost', 1999;5:25–27. EDN NVBNEP (in Russ.).
12. Reshetnik E. I., Sharipova T. V., Maksimyuk V. A. Possibility of application chick-pea flour in the production of meat-vegetable prepared foods for elderly nutrition. Dal'nevostochnyy agrarnyy vestnik, 2014;1(29):48–51. EDN TMWSRL (in Russ.).
13. Miftakhutdinova E. A., Tikhonov S. L., Tikhonova N. V., Pestova I. G., Pishchikov G. B., Popova D. G. [et al.]. Meat pate for gerodietic nutrition for active lifestyle. Polzunovskiy vestnik, 2020;2:70–74. https://doi.org/10.25712/ASTU.2072-8921.2020.02.013. EDN SJTHKM (in Russ.).
14. Miftahutdinova E. A., Tikhonov S. L., Tikhonova N. V. Development of lithiumcontaining feed additive and its use for fortification of chicken broilers meat and by-products. Theory and Practice of Meat Processing, 2020;5;1:27–31. https://doi.org/10.21323/2414-438X-2020-5-1-27-31.
15. Loch J. I., Bonarek P., Tworzydlo M., Polit A., Hawro B., Lach A. [et al.]. Engineered b-lactoglobulin produced in E. coli: purification, biophysical and structural characterization. Molecular Biotechnology, 2016;58;10:605–618. https://doi.org/10.1007/s12033-016-9960-z.
16. Jensen H. B., Holland J. W., Poulsen N. A., Larsen L. B. Milk protein genetic variants and isoforms identified in bovine milk representing extremes in coagulation properties. Journal of Dairy Science, 2012;95;6:2891–2903. https://doi.org/10.3168/jds.2012-5346.
17. Kато T., Lee R. T. GDF-11 as a potential cardiac pro-angiogenic factor. JACC. Basic to Translational Science, 2023;8;6:636–637. https://doi.org/10.1016/j.jacbts.2023.04.003.
18. Lian J., Walker R. G., D’Amico A., Vujic A., Mills M. J., Messemer K. A. [et al.]. Functional substitutions of amino acids that differ between GDF11 and GDF8 impact skeletal development and skeletal muscle. Life Science Alliance, 2023;6;3:e202201662. https://doi.org/10.26508/lsa.202201662.
19. Harper S. C., Johnson J., Borghetti G., Zhao H., Wang T., Wallner M. [et al.]. GDF11 decreases pressure overload-induced hypertrophy, but can cause severe cachexia and premature death. Circulation Research, 2018;123;11:1220–1231. https://doi.org/10.1161/CIRCRESAHA.118.312955.
20. Cooper B. M., Iegre J., O' Donovan D. H., Halvarsson M. Ö., Spring D. R. Peptides as a platform for targeted therapeutics for cancer: peptide-drug conjugates (PDCs). Chemical Society reviews, 2021;50;3:1480–1494. https://doi.org/10.1039/d0cs00556h.
21. Di L. Strategic approaches to optimizing peptide ADME properties. The AAPS Journal, 2015;17;1:134–143. https://doi.org/10.1208/s12248-014-9687-3.
22. Wu H., Huang J. Optimization of protein and peptide drugs based on the mechanisms of kidney clearance. Protein and Peptide Letters, 2018;25;6:514–521. https://doi.org/10.2174/0929866525666180530122835.