Z. R. Khasanshina, A. V. Kazakova, S. A. Ishchuk, I. A. Kornakov, S. S. Timofeev, V. I. Shmurak, R. V. Drai
{"title":"Development of a Technology for Producing Therapeutic Peptides Weighting Less Than 5 kDa in a Bacterial Expression System","authors":"Z. R. Khasanshina, A. V. Kazakova, S. A. Ishchuk, I. A. Kornakov, S. S. Timofeev, V. I. Shmurak, R. V. Drai","doi":"10.1134/S1990750824600341","DOIUrl":null,"url":null,"abstract":"<p>Therapeutic peptides are an interesting class of pharmaceutical compounds that are structurally intermediate between small molecules and proteins but biochemically and therapeutically distinct from both. Currently, peptide-based drugs are used to treat immunological diseases, infections, and endocrinological and oncological diseases. The expression of therapeutic peptides in bacterial systems, for example, in <i>E. coli</i>, has a large number of advantages, such as high specific productivity, low production costs, great optimization potential, and good knowledge of the process. However, the creation of bacterial producer strains expressing therapeutic peptides with a molecular weight of less than 5 kDa is an extremely complex and time-consuming task due to the rapid degradation of the product by cell proteolytic enzymes or the high cytotoxicity of the target product. To solve such problems, the peptide is expressed in the form of tandem repeats or as a hybrid protein fused with another large protein, for example, SUMO. In this work, four peptide antigens differing in physicochemical properties were purified. The peptides were synthesized in soluble form as precursor proteins consisting of an N-terminal polyhistidine tag (His 6 tag), a SUMO fragment, and the amino acid sequence of the peptide. Hybrids were isolated from bacterial cells by disintegrating biomass under high pressure. The proteins were then purified by metal affinity chromatography using Ni-NTA agarose and enzymatically hydrolyzed with a highly specific SUMO protease. The resulting peptides were purified using metal affinity chromatography and final high-performance reverse phase chromatography on an SP-100-C8-PK sorbent. Next, the peptides were freeze-dried. As a result of work using the general technology, four peptides with different physicochemical properties were obtained. The purity by RP-HPLC was greater than 95%, and the mass of all peptides and fusion proteins was confirmed by HPLC-MS. The authenticity of the peptides was additionally confirmed by the ELISA method.</p>","PeriodicalId":485,"journal":{"name":"Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry","volume":"19 1","pages":"41 - 51"},"PeriodicalIF":0.4000,"publicationDate":"2025-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry","FirstCategoryId":"2","ListUrlMain":"https://link.springer.com/article/10.1134/S1990750824600341","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Therapeutic peptides are an interesting class of pharmaceutical compounds that are structurally intermediate between small molecules and proteins but biochemically and therapeutically distinct from both. Currently, peptide-based drugs are used to treat immunological diseases, infections, and endocrinological and oncological diseases. The expression of therapeutic peptides in bacterial systems, for example, in E. coli, has a large number of advantages, such as high specific productivity, low production costs, great optimization potential, and good knowledge of the process. However, the creation of bacterial producer strains expressing therapeutic peptides with a molecular weight of less than 5 kDa is an extremely complex and time-consuming task due to the rapid degradation of the product by cell proteolytic enzymes or the high cytotoxicity of the target product. To solve such problems, the peptide is expressed in the form of tandem repeats or as a hybrid protein fused with another large protein, for example, SUMO. In this work, four peptide antigens differing in physicochemical properties were purified. The peptides were synthesized in soluble form as precursor proteins consisting of an N-terminal polyhistidine tag (His 6 tag), a SUMO fragment, and the amino acid sequence of the peptide. Hybrids were isolated from bacterial cells by disintegrating biomass under high pressure. The proteins were then purified by metal affinity chromatography using Ni-NTA agarose and enzymatically hydrolyzed with a highly specific SUMO protease. The resulting peptides were purified using metal affinity chromatography and final high-performance reverse phase chromatography on an SP-100-C8-PK sorbent. Next, the peptides were freeze-dried. As a result of work using the general technology, four peptides with different physicochemical properties were obtained. The purity by RP-HPLC was greater than 95%, and the mass of all peptides and fusion proteins was confirmed by HPLC-MS. The authenticity of the peptides was additionally confirmed by the ELISA method.
期刊介绍:
Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry covers all major aspects of biomedical chemistry and related areas, including proteomics and molecular biology of (patho)physiological processes, biochemistry, neurochemistry, immunochemistry and clinical chemistry, bioinformatics, gene therapy, drug design and delivery, biochemical pharmacology, introduction and advertisement of new (biochemical) methods into experimental and clinical medicine. The journal also publishes review articles. All issues of the journal usually contain solicited reviews.