Preparative Production and Purification of Recombinant Human Cyclophilin Aстатья
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Аннотация:In this work, we developed the method of preparative production of recombinant human cyclophilin A (rhCypA)in Escherichia coli. The fulllength cDNA encoding the gene of human CypA (CYPA) was amplified by RTPCR from thetotal RNA of human T cell lymphoma Jurkat. The nucleotide sequence of CYPA was optimized to provide highly effectivetranslation in E. coli. Recombinant CYPA DNA was cloned into the pET22b(+) vector, and the resulted expression plasmidwas used to transform E. coli strain BL21(DE3)Gold. The recombinant producer strain of E. coli produced soluble rhCypAin the bacterial cytoplasm. The synthesis efficiency of rhCypA was up to 50% of the total cell protein allowing to producerhCypA in the amount of 1 g per liter of the culture. We also developed the method for rhCypA purification, consisting of asinglestep tandem anion exchange chromatography on DEAE and QSepharose columns. The protein purity was 95%according to electrophoresis (SDSPAGE), and its contamination with endotoxin did not exceed 0.05 ng per 1 mg of theprotein 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 stimulationof migration of mouse bone marrow hematopoietic stem cells in vivo. The generated producer strain of E. coli is a superproducer and could be used for largescale experimental studies of rhCypA and in its preclinical and clinical trials as a drug