Continuous-exchange cell-free protein-synthesizing system: synthesis of HIV-1 antigen Nef.
HIV-1 antigen Nef, Green Fluorescent Protein (GFP), and the fusion protein GFP-Nef ("Green Fluorescent Nef") were synthesized in bacterial cell-free system with continuous supply of substrates and continuous removal of low-molecular-weight products through a dialysis membrane during incubation, the so-called continuous-exchange cell-free (CECF) system. The identity of synthesized proteins was confirmed by electrophoretic mobility, Western blotting, and GFP fluorescence. The system produced several nanomoles (hundreds of microg) of each protein per ml of the reaction mixture. Construction of GFP-fused proteins is considered as a general strategy for visualization and monitoring of cell-free production of the proteins that have no easily testable functions.
Chekulayeva
M N
MN
Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.
Kurnasov
O V
OV
Shirokov
V A
VA
Spirin
A S
AS
D000956:Antigens, Viral / Q000096:biosynthesis* / Q000235:genetics; D015153:Blotting, Western; D003956:Dialysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D015693:Gene Products, nef / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D054311:nef Gene Products, Human Immunodeficiency Virus
D000956:Antigens, Viral; D015693:Gene Products, nef; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins