title: Synthesis of a eukaryotic virus protein in a prokaryotic viral-cell system: production of the adenovirus type 2 fiber shaft fragment by a tightly regulated T7POL-M13 expression system.
abstract: The use of recombinant technology for the production of proteins of interest in biotechnology and medicine has grown immensely during the last decade. A major problem often encountered is the degradation of the recombinant product by host cell proteases. We developed a novel system based on the cloning and expression of an inducible phage T7 RNA polymerase into the main intergenic region of the phage M13-KO7. After infection of permissive bacterial strains with the engineered phage, the polymerase gene is transcribed, subsequently translated and gene fragments cloned under T7 promoter sequences are then transcribed. For the evaluation of this system, the gene encoding the shaft fragment of the adenovirus type 2 fiber was cloned into a pET 3a-based expression vector. Expression was demonstrated in a BL21(DE3) strain (containing one copy of the T7 RNA polymerase gene) and also in several F pili-containing bacterial strains only after infection with the proper bacteriophage. Several important parameters for heterologous gene expression in Escherichia coli were investigated. Different bacterial strains were evaluated for the production of the recombinant protein, following: the expression levels, the growth rates and the stability of the plasmid vector at different time intervals after induction. It was observed that the expression levels as well as division rates and plasmid stability differed between the different bacterial strains. The best expression levels were obtained when using the E. coli Top IOF' strain. Degradation was only observed in BL21(DE3) cells after 6 h of induction, whereas none of the F'-containing cells were shown to degrade the recombinant protein during the time of expression. This system, based on the T7 pol-M13 bacteriophage, was shown to be very tightly regulated for most of the bacterial strains evaluated with no expression before induction of the T7 RNA polymerase.
authors.affiliation: Immunotechnology and Diagnosis Division, Center for Genetic Engineering and Biotechnology (CIGB), Havanna, Cuba.
authors.forename: M
authors.initials: M
authors.lastname: Dueñas
authors.forename: E
authors.initials: E
authors.lastname: Rodríguez
authors.forename: N
authors.initials: N
authors.lastname: Nilsson
authors.forename: E
authors.initials: E
authors.lastname: Everitt
authors.forename: C
authors.initials: C
authors.lastname: Borrebaeck
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics*; D017104:Bacteriophage M13 / Q000235:genetics; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics*; D036022:Capsid Proteins; D003001:Cloning, Molecular / Q000379:methods*; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli; D005057:Eukaryotic Cells; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011387:Prokaryotic Cells; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C018315:hexon capsid protein, Adenovirus; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases