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

The use of recombinant technology for the production of proteins of interes t in biotechnology and medicine has grown immensely during the last decade. A major problem often encountered is the degradation of the recombinant pr oduct by host cell proteases. We developed a novel system based on the clon ing and expression of an inducible phage T7 RNA polymerase into the main in tergenic region of the phage M13-KO7. After infection of permissive bacteri al strains with the engineered phage, the polymerase gene is transcribed, s ubsequently translated and gene fragments cloned under T7 promoter sequence s are then transcribed. For the evaluation of this system, the gene encodin g the shaft fragment of the adenovirus type 2 fiber was cloned into a pET 3 a-based expression vector. Expression was demonstrated in a BL21(DE3) strai n (containing one copy of the T7 RNA polymerase gene) and also in several F pill-containing bacterial strains only after infection with the proper bac teriophage. Several important parameters for heterologous gene expression i n Escherichia coli were investigated. Different bacterial strains were eval uated for the production of the recombinant protein, following: the express ion levels, the growth rates and the stability of the plasmid vector at dif ferent 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 10F' strain. Degradation was only observed in BL21(D E3) cells after 6 h of induction, whereas none of the F'-containing cells w ere 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 t ightly regulated for most of the bacterial strains evaluated with no expres sion before induction of the T7 RNA polymerase. (C) 1999 Elsevier Science B .V. All rights reserved.