Preparation and Characterization of a New Mutant Homolog of the Chemotaxis Protein CheY from the Anaerobic Hyperthermophilic Microorganism Thermotoga Naphthophila

Abstract—: Using genetic engineering methods we have developed expression vectors for synthesis of recombinant proteins TnaCheY and TnaCheY-mut, the homologues of the chemotaxis protein CheY from the hyperthermophilic organism Thermotoga naphthophila in Escherichia coli BL21(DE3) cells. The cultivation conditions of transformed cell strains were optimized. The influence of episomal expression of the heterologous chemotaxis protein CheY on growth kinetics parameters of the culture of mesophilic bacteria E. coli was investigated. The optimal purification flowchart of the obtained proteins using thermolysis has been proposed. Based on the data obtained, we discuss potential areas of application of recombinant variants of the CheY thermostable chemotactic protein. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment of the episomal expression of such proteins to control the cultivation and production time of pharmaceutically and industrially valuable metabolites due to the impact on some stages of the bacterial chemotaxis has been experimentally proven. © 2019, Pleiades Publishing, Ltd

Preparation and characterization of a new mutant homolog of chemotaxis protein CheY from anaerobic hyperthermophilic microorganism Thermotoga naphthophila [Poluchenie i kharakteristika novogo mutantnogo gomologa khemotaksisnogo belka CheY iz gipertermofil'nogo anaérobnogo mikroorganizma Thermotoga naphthophila]

Using genetic engineering methods the expression vectors structures have been designed to produce recombinant proteins TnaCheY and Tna CheY-mut, the homologues of the chemotaxis protein CheY from the hyperthermophilic organism Thermotoga naphthophila in Escherichia coli BL21(DE3) cells. The cultivation conditions of transformed strains were optimized. The influence of episomal expression of the heterologous chemotaxis protein CheY on growth kinetics parameters of the culture of mesophilic bacteria E. coli was studied. The optimal purification flowchart of the obtained proteins using thermolysis is proposed. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment the episomal expression of such proteins to control the cultivation and production time of pharmaceutically and industrially valuable metabolites due to the impact on some stages of the bacterial chemotaxis is experimentally proved.S ispol'zovaniem genno-inzhenernykh metodov skonstruirovany ékspressionnye vektornye konstruktsii, obespechivaiushchie éffektivnuiu produktsiiu rekombinantnykh belkov TnaCheY i TnaCheY-mut – gomologov khemotaksisnogo belka CheY gipertermofil'nogo mikroorganizma Thermotoga naphthophila – v kletkakh Escherichia coli BL21(DE3). Optimizirovany usloviia kul'tivirovaniia transformirovannykh shtammov. Izucheno vliianie épisomal'noĭ ékspressii geterologichnogo khemotaksisnogo belka CheY na parametry kinetiki rosta kul'tury mezofil'nogo mikroorganizma E. coli. Predlozhena optimal'naia skhema ochistki poluchennykh belkov s ispol'zovaniem termolizisa. Na osnove poluchennykh dannykh v stat'e rassmotreny potentsial'nye oblasti primeneniia rekombinantnykh variantov termostabil'nogo khemotaksisnogo belka CheY. Na primere kletok laboratornogo shtamma E. coli BL21(DE3), éksperimental'no obosnovana vozmozhnost' ispol'zovaniia épisomal'noĭ ékspressii podobnykh belkov dlia upravleniia vremenem kul'tivirovaniia i produktsii farmatsevticheski i promyshlenno tsennykh metabolitov za schet vozdeĭstviia na otdel'nye étapy khemotaksisa bakteriĭ