Engineering hyperexpression of bacteriophage Mu C protein by removal of secondary structure at the translation initiation region

Abstract

The structure at the translation initiation region (TIR) of mRNA has pronounced regulatory effects on gene expression. Our attempts to overexpress the C gene of bacteriophage Mu in a variety of expression vectors resulted in low yields of protein. Analysis of Mu C mRNA shows the potential to form a secondary structure involving a ribosome binding site and AUG codon. We haveengineered the overproduction of the protein using a PCR-aided cloning approach to remove the sequences involved in the formation of this secondary structure. The overexpressing clone, underthe control of T7 gene 10 promoter in a T7 expression system yielded > 30% of total cellprotein. The difference in mRNA structure between expressing and non-expressing clones was confirmed by electrophoretic 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. DNA 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×l0¹² M^-1.