在细菌表达系统中生产重量小于5kda的治疗性肽技术的发展

IF 0.4 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY
Z. R. Khasanshina, A. V. Kazakova, S. A. Ishchuk, I. A. Kornakov, S. S. Timofeev, V. I. Shmurak, R. V. Drai
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引用次数: 0

摘要

治疗肽是一类有趣的药物化合物,在结构上介于小分子和蛋白质之间,但在生物化学和治疗上与两者不同。目前,肽类药物主要用于免疫疾病、感染、内分泌和肿瘤疾病的治疗。在细菌系统中表达治疗肽,例如在大肠杆菌中,具有许多优点,例如高比产率,低生产成本,巨大的优化潜力以及对过程的良好了解。然而,由于细胞蛋白水解酶的快速降解或目标产物的高细胞毒性,制造表达分子量小于5kda的治疗肽的细菌生产菌株是一项极其复杂和耗时的任务。为了解决这些问题,肽以串联重复序列的形式表达,或者作为与另一个大蛋白融合的杂交蛋白,例如SUMO。在这项工作中,纯化了四种物理化学性质不同的肽抗原。这些肽以可溶性形式作为前体蛋白合成,由一个n端多组氨酸标签(His 6标签)、一个SUMO片段和肽的氨基酸序列组成。杂种是通过高压分解生物量从细菌细胞中分离出来的。然后用Ni-NTA琼脂糖通过金属亲和层析纯化蛋白质,并用高度特异性的SUMO蛋白酶进行酶解。通过金属亲和层析和SP-100-C8-PK吸附剂的高效反相层析纯化所得肽。接下来,将多肽冷冻干燥。采用通用技术,得到了四种具有不同理化性质的多肽。反相高效液相色谱法纯度大于95%,高效液相色谱-质谱法证实了所有肽段和融合蛋白的质量。用ELISA法进一步证实了肽段的真实性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Development of a Technology for Producing Therapeutic Peptides Weighting Less Than 5 kDa in a Bacterial Expression System

Development of a Technology for Producing Therapeutic Peptides Weighting Less Than 5 kDa in a Bacterial Expression System

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.

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来源期刊
CiteScore
1.10
自引率
0.00%
发文量
31
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.
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