High-level autoenhanced expression of a single-copy gene in Escherichia coli: overproduction of bacteriophage T7 protein kinase directed by T7 late genetic elements

Bacteriophage T7 early gene 0.7 assists phage growth under suboptimal condi tions ('helper' function). Whereas the C-terminal one-third of the encoded protein participates in host transcription shutoff, the N-terminal two-thir ds harbours a protein kinase ('PK') activity with broad specificity. Howeve r, how this activity relates to helper function is unclear. Here, a truncat ed gene 0.7 encoding PK was fused to an IPTG-inducible T7 late promoter and to a translation initiation region from a T7 late gene, and inserted into the chromosome of an E. coli strain expressing T7 RNA polymerase. After ind uction, total protein synthesis remains unchanged but with over 40% devoted to PK synthesis, an amazing figure for the expression of a single-copy gen e. Mutations abolishing PK activity reduce this expression by 3-fold. Thus, PK activity stimulates PK expression when the latter is controlled by T7 l ate genetic elements. Further experiments show that stimulation occurs at b oth transcriptional and post-transcriptional levels. The helper function ma y therefore correspond to a PK-mediated stimulation of late expression, the mechanism of which is discussed. The possibility of exploiting the PK acti vity fur improving E. coli expression systems is also considered. (C) 2001 Elsevier Science B.V. All rights reserved.