N. N. Blokhin National Medical Research Center of Oncology, the Ministry of Health of the Russian Federation, Moscow, 115478, Russia.
State Research Center of Applied Microbiology and Biotechnology, Obolensk, Moscow Region, 142279, Russia.
Biochemistry (Mosc). 2022 Mar;87(3):259-268. doi: 10.1134/S0006297922030063.
In this work, we developed the method of preparative production of recombinant human cyclophilin A (rhCypA) in Escherichia coli. The full-length cDNA encoding the gene of human CypA (CYPA) was amplified by RT-PCR from the total RNA of human T cell lymphoma Jurkat. The nucleotide sequence of CYPA was optimized to provide highly effective translation in E. coli. Recombinant CYPA DNA was cloned into the pET22b(+) vector, and the resulted expression plasmid was used to transform E. coli strain BL21(DE3)Gold. The recombinant producer strain of E. coli produced soluble rhCypA in the bacterial cytoplasm. The synthesis efficiency of rhCypA was up to 50% of the total cell protein allowing to produce rhCypA in the amount of 1 g per liter of the culture. We also developed the method for rhCypA purification, consisting of a single-step tandem anion exchange chromatography on DEAE- and Q-Sepharose columns. The protein purity was 95% according to electrophoresis (SDS-PAGE), and its contamination with endotoxin did not exceed 0.05 ng per 1 mg of the protein that met the requirements of European pharmacopoeia for injectable preparations. The produced recombinant protein exhibited functional features of native CypA, i.e., isomerase activity and chemokine activity as assessed by stimulation of migration of mouse bone marrow hematopoietic stem cells in vivo. The generated producer strain of E. coli is a super-producer and could be used for large-scale experimental studies of rhCypA and in its preclinical and clinical trials as a drug.
在这项工作中，我们开发了在大肠杆菌中制备生产重组人亲环素 A（rhCypA）的方法。通过 RT-PCR 从人 T 细胞淋巴瘤 Jurkat 的总 RNA 扩增编码人 CypA（CYPA）基因的全长 cDNA。优化了 CYPA 的核苷酸序列，以在大肠杆菌中提供高效翻译。将重组 CYPA DNA 克隆到 pET22b(+)载体中，并将所得表达质粒用于转化大肠杆菌菌株 BL21(DE3)Gold。大肠杆菌的重组生产菌株在细菌细胞质中产生可溶性 rhCypA。rhCypA 的合成效率高达总细胞蛋白的 50%，允许在 1 升培养物中产生 1 克 rhCypA。我们还开发了 rhCypA 纯化方法，包括在 DEAE-和 Q-Sepharose 柱上单步串联阴离子交换层析。根据电泳（SDS-PAGE），蛋白质纯度为 95%，其与内毒素的污染不超过 0.05ng/每毫克蛋白质，符合欧洲药典对可注射制剂的要求。所产生的重组蛋白表现出天然 CypA 的功能特征，即通过体内刺激小鼠骨髓造血干细胞的迁移来评估的异构酶活性和趋化因子活性。产生的大肠杆菌生产菌株是一种超级生产菌株，可用于 rhCypA 的大规模实验研究及其作为药物的临床前和临床试验。
