Improved method for expression and isolation of the Mycoplasma hominis arginine deiminase from the recombinant strain of Escherichia coli.
Arginine deiminase is a promising anticancer drug active against melanoma, hepatocarcinoma and other tumors. Recombinant strains of Escherichia coli that express arginine deiminase from pathogenic bacteria Mycoplasma have been developed. However, production costs of heterologous arginine deiminase are high due to use of an expensive inducer and extraction buffer, as well as using diluted culture for enzyme induction. We report on a new advanced protocol for Mycoplasma hominis arginine deiminase expression, extraction and renaturation. The main improvements include manipulation with dense suspensions of E. coli, use of lactose instead of isopropyl β-D-1-thiogalactopyranoside as an inducer and a cheaper but not less efficient buffer for solubilization of arginine deiminase inclusion bodies. In addition, supplementation of the storage culture medium with glucose and substrate (arginine) significantly stabilized the recombinant arginine deiminase producer. Homogenous preparations of recombinant arginine deiminase were obtained using anion-exchange and hydrophobic chromatography. The purified enzyme retained a specific activity of 30-34 U/mg for 12 months when stored at 4°C in 20 mM sodium phosphate buffer pH 7.2 containing 1 M NaCl.
Institute of Cell Biology, NAS of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine.
Lyubov R
LR
Fayura
Yuriy R
YR
Boretsky
Yuriy V
YV
Pynyaha
Denys N
DN
Wheatley
Andriy A
AA
Sibirny
D000970:Antineoplastic Agents / Q000494:pharmacology; D001709:Biotechnology; D003001:Cloning, Molecular; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006867:Hydrolases / Q000191:economics / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002479:Inclusion Bodies; D007785:Lactose / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D019535:Mycoplasma hominis / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D000970:Antineoplastic Agents; D003470:Culture Media; D011994:Recombinant Proteins; D006867:Hydrolases; C019723:arginine deiminase; D007785:Lactose
Arginine deiminase; Expression; Producer stabilization; Purification