title: 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 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.
authors: Centre for Genetic Engineering, Indian Institute of Science, Bangalore.
authors: V
authors: V
authors: Ramesh
authors: A
authors: A
authors: De
authors: V
authors: V
authors: Nagaraja
mesh_terms: D010583:Bacteriophage mu; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D011485:Protein Binding; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D015534:Trans-Activators / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C090591:C protein, Bacteriophage Mu; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D014764:Viral Proteins