Investigation of biosynthesis of human lymphotoxin in cells of recombinant Escherichia coli strain.
The effect of cultivation conditions on the biosynthesis of human lymphotoxin in recombinant Escherichia coli SG20050/pLT21 strain was studied. Cells of the producing strain were grown in Luria broth containing chloramphenicol. The highest biomass yield of the recombinant strain and plasmid DNA stability were observed under these conditions. To enhance the level of lymphotoxin production an inoculate containing freshly obtained or frozen with glycerol transformants of the producing strain were used, and the cultivation process was performed at 32 degrees C. As a result, lymphotoxin was synthesized in a soluble form without the formation of inclusion bodies. A study of the protein synthesis dynamics during the cultivation of E. coli at 32 degrees C showed that the highest lymphotoxin activity was observed during the exponential growth phase, being maximal at the end of the exponential phase and at the beginning of the stationary phase. The set of indicated methods allowed us to maximize and stabilize the production of lymphotoxin in a biologically active form with a final yield of 18-20% from cell protein.
Denisov
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AA
State Research Center for Applied Microbiology, Obolensk, Moscow Region, 142279, Russia.
Nikolaeva
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OG
D002455:Cell Division / Q000502:physiology; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D018628:Gene Dosage; D006801:Humans; D008233:Lymphotoxin-alpha / Q000096:biosynthesis*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification*; D013696:Temperature; D014170:Transformation, Genetic / Q000235:genetics
D003470:Culture Media; D008233:Lymphotoxin-alpha; D011994:Recombinant Proteins