ENGINEERING HYPEREXPRESSION OF BACTERIOPHAGE-MU C-PROTEIN BY REMOVAL OF SECONDARY STRUCTURE AT THE TRANSLATION INITIATION REGION

The structure at the translation initiation region (TIR) of mRNA has p ronounced regulatory effects on gene expression. Our attempts to overe xpress the C gene of bacteriophage Mu in a variety of expression vecto rs resulted in low yields of protein. Analysis of Mu C mRNA shows the potential to form a secondary structure involving a ribosome binding s ite and AUG codon. We have engineered the overproduction of the protei n using a PCR-aided cloning approach to remove the sequences involved in the formation of this secondary structure. The overexpressing clone , under the control of T7 gene 10 promoter in a T7 expression system y ielded > 30% of total cell protein. The difference in mRNA structure b etween expressing and non-expressing clones was confirmed by electroph oretic analysis of run-off transcripts. The overexpressed protein was purified in a single step by site-specific DNA affinity chromatography . The purified recombinant protein was active in band shift assays. DN A binding activity required Mg2+ and was weak in the presence of Mn2+. Cd2+ or Zn2+ could not support DNA binding. Under optimal conditions, the equilibrium binding constant (K-app) was determined to be 2 x 10( 12) M(-1).