[Optimization of induction and purification of HIV-1 Gag protein in Escherichia coli expression system].
To investigate the effects of induction temperature on the expression product and the impact of urea concentration on the purification, HIV-1 Gag inclusion bodies from E. coli induced at 30 degrees C (IB30) and 37 degrees C (IB37) were dissolved with urea of different concentrations. The solubility and yield of refolding were compared. IB30 were dissolved with 2 mol/L and 8 mol/L urea, and then purified with chromatography. IB30 were found easier to be solubilized in low concentration of urea and easier to be refolded than IB37. Furthermore, compared to the IB30 dissolved in 8 mol/L urea, Gag protein solubilized in 2 mol/L urea was purified to higher purity with gel filtration (GF) and ion exchange (IEX) chromatography. Gag inclusion body induced at lower temperature may contain more protein with native-like or reversibly-denatured structures, and solubilization in the presence of low concentrations of urea can help to retain these structures. This study has provided new insights into the purification of proteins from inclusion bodies.
State Key Laboratory for Infectious Diseases Prevention and Control, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100050, China.
Jingjing
J
Fu
Jing
J
Sun
Pei
P
Chen
Zhu
Z
Huo
Yanling
Y
Hao
Yong
Y
Liu
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D013696:Temperature; D014508:Urea / Q000737:chemistry; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D014508:Urea