The regulatory elements of araBAD operon, contrary to lac-based expression systems, afford hypersynthesis of murine, and human interferons in Escherichia coli.
The overexpression of four different interferons, i.e., murine interferon alpha1 and human interferons alpha1, alpha 8, and alpha 21 was challenged in Escherichia coli. Synthetic genes coding for these interferons were designed, assembled, and cloned into the vector pET9a (using the NdeI and BamHI sites), placing interferon expression under the control of phage T7 promoter. Despite an intensive screening for optimal culture conditions, no interferon synthesis was observed using overexpression systems based on the regulatory elements of lac operon (e.g., in E. coli BL21DE3). On the contrary, high levels of interferon expression were detected in E. coli BL21AI, which chromosome contains the gene coding for phage T7 RNA polymerase under the control of the araBAD promoter. To analyze the reasons of this striking difference, the molecular events associated with the lack of interferon expression in E. coli BL21DE3 were studied, and murine interferon alpha1 was chosen as a model system. Surprisingly, it was observed that this interferon represses the synthesis of T7 RNA polymerase in E. coli BL21DE3 and, in particular, the expression of lac operon. In fact, by determining beta-galactosidase activity in E. coli BL21AI, a significantly lower LacZ activity was observed in cells induced to interferon synthesis.
Stefan
Alessandra
A
Department of Industrial Chemistry, University of Bologna, Bologna 40136, Italy.
Alfarano
Pietro
P
Merulla
Davide
D
Mattana
Paolo
P
Rolli
Eleonora
E
Mangino
Pierluigi
P
Masotti
Lanfranco
L
Hochkoeppler
Alejandro
A
D000818:Animals; D001089:Arabinose / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007370:Interferon Type I / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D007763:Lac Operon / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014764:Viral Proteins / Q000235:genetics
D007370:Interferon Type I; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014764:Viral Proteins; D001089:Arabinose; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases