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Engineering hyperexpression of bacteriophage Mu C protein by removal of secondary structure at the translation
1Centre for Genetic Engineering, Indian Institute of Science, Bangalore.
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.
Insights
Overcoming mRNA secondary structure issues allowed high-level production of bacteriophage Mu C protein. This engineered protein was purified and confirmed to be active in DNA binding assays.
Area of Science:
- Molecular Biology
- Protein Expression
- Biochemistry
Background:
- The translation initiation region (TIR) of mRNA significantly regulates gene expression.
- Attempts to overexpress bacteriophage Mu C gene resulted in low protein yields due to mRNA secondary structures.
Purpose of the Study:
- To engineer the overproduction of bacteriophage Mu C protein.
- To overcome challenges posed by mRNA secondary structures in the TIR.
Main Methods:
- PCR-aided cloning to remove mRNA secondary structure-forming sequences.
- Utilizing a T7 expression system with a T7 gene 10 promoter.
- Site-specific DNA affinity chromatography for protein purification.
- Electrophoretic analysis of run-off transcripts to confirm mRNA structure differences.
Main Results:
- Engineered clone yielded over 30% of total cell protein.
- Confirmed mRNA structural differences between expressing and non-expressing clones.
- Purified recombinant protein showed activity in band shift assays.
- Determined optimal DNA binding conditions and equilibrium binding constant (Kapp = 2 x 10(12) M-1).
Conclusions:
- Successfully engineered high-level production of active bacteriophage Mu C protein.
- Demonstrated the critical role of TIR mRNA structure in protein expression.
- Characterized the DNA binding activity and requirements of the purified protein.