ISSN Print 2500–1094
ISSN Online 2542–1204
BIOMEDICAL JOURNAL OF PIROGOV UNIVERSITY (MOSCOW, RUSSIA)
ORIGINAL RESEARCH
Preparation of a recombinant ribonuclease inhibitor in E. coli for use in mRNA synthesis in vitro
About authors
1 Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russia
2 Sirius University of Science and Technology, Sirius, Sochi, Russia
Correspondence should be addressed: Maxim O. Nagornykh
Prospekt Nauki, 5, Pushchino, 142290, Russia; moc.liamg@rennabred
About paper
Funding: the study was financially supported in the context of the program of Ministry of higher education and science of the Russian Federation (agreement #075-10-2021-113, unique project number RF----193021X0001).
Author contribution: Zakharov MV — selection of conditions of production of recombinant proteins, production experiments in different E. coli strains; Zagoskin AA — chromatographic clearing of recombinant proteins; Nagornykh MO — conceptualization, design of genetic constructs, article authoring; Ivanov RA — general management.
Received: 2023-11-16
Accepted: 2023-12-17
Published online: 2023-12-31
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Fig. 1. Maps of vectors for production of ribonuclease inhibitor with the fusion polypeptides that increase solubility of recombinant proteins in E. coli; A — variants based on the pET28a vector, T7 promoter, B — variants based on the pSol vector, PRha promoter. RNH1 — gene encoding the human ribonuclease inhibitor; hPP — gene encoding one of the fusion polypeptides (MBP, TIG, TSF, FH8, PpiB, YrhB, SUMO, TRX, GST). pET28a vector backbone contains the kanamycin resistance gene, pBR322 replication origin, and the sequence encoding the lacI repressor. pSol vector backbone contains the kanamycin resistance gene, pUC replication origin, the rhamnose-induced PRha promoter, and the transcription terminator
Fig. 2. Effect of fusion polypeptides on solubility of ribonuclease inhibitor, production in E. coli BL21 (DE3) strain at 16 °C; A — variants based on the pET28a vector, T7 promoter, B — variants based on the pSol vector, PRha promoter. IS is the relative level of insoluble fraction, shown in red. SO is the the level produced soluble ribonuclease inhibitor, shown in blue. Density RU are densitometry of PAAG results shown in relative units
Fig. 3. Selection of the E. coli strain optimal for production of the recombinant ribonuclease inhibitor in soluble form, using vectors encoding a chimeric protein consisting of combinations of a ribonuclease inhibitor and two fusion polypeptides (MBP, TIG). IS is the relative level of insoluble fraction, shown in red. SO is the the level produced soluble ribonuclease inhibitor, shown in blue. Density RU are densitometry of PAAG results shown in relative units
Fig. 4. Effect of additional gene co-expression, cellular chaperones dnaK, dnaJ, grpE (shown as KJE), groES and groEL (shown as ELS), on production of the recombinant ribonuclease inhibitor in the KRX E. coli strain at 16 °C. IS is the relative level of insoluble fraction, shown in red. SO is the the level produced soluble ribonuclease inhibitor, shown in blue. Density RU are densitometry of PAAG results shown in relative units
