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 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.
Ramesh
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Centre for Genetic Engineering, Indian Institute of Science, Bangalore.
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Nagaraja
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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
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