Ramesh, V and De, Amitabha and Nagaraja, V (1994) Engineering hyperexpression of bacteriophage Mu C protein by removal of secondary structure at the translation initiation region. In: Protein Engineering, 7 (8). pp. 1053-1057.
Restricted to Registered users only
Download (862Kb) | Request a copy
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 have engineered 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, under the control of T7 gene 10 promoter in a T7 expression system yielded > 30% of total cell protein. 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 (Kapp) was determined to be 2 x 10(12) M-1.
|Item Type:||Journal Article|
|Additional Information:||Copyright for this article belongs to Oxford University Press.|
|Keywords:||mRNA secondary structure;Mu C;overproduction|
|Department/Centre:||Division of Biological Sciences > Microbiology & Cell Biology|
|Date Deposited:||01 Sep 2004|
|Last Modified:||19 Sep 2010 04:14|
Actions (login required)