The use of N-terminal peptide fusions to stimulate recombinant gene expression in Escherichia coli

Abstract

Previous studies by the Microbiology and Molecular Genetics (MOLGEN) research group at NTNU have investigated how N-terminal peptide fusions influence gene expression levels. The present study seeks to expand these efforts to reveal possible mechanisms that promote increased expression by application of peptide fusions. To meet this aim, novel peptide fusions of increasing lengths were constructed using the celB gene encoding phosphoglucomutase as a template. This was conducted to establish how the length of celB peptide fusions influences gene expression. Furthermore, the effect of 5´ terminal end mRNA secondary structures on gene expression was investigated. This was achieved by introduction of synonymous mutations to the celB fusions to possibly stabilize or eliminate secondary structures in the transcripts. To establish if the peptide fusions influence gene expression in a universal manner, three different proteins of medical importance were adopted as model proteins: IFN-α2b, GM-CSF and scFv-phOx-AP The results gained from this study suggest that celB fusion partners act to increase expression in a manner that positively correlates with the length of the celB fusion sequence. Furthermore, it has been demonstrated that mRNA transcript levels do not fully correlate with levels of translated protein. Hence, it is assumed that peptide fusions influence both transcription/mRNA stability and translational efficiency. Two different mechanisms have been suggested to explain the stimulative effect of celB fusion partners on gene expression: increased ribosomal recruitment and optimized distribution of ribosomes along the transcript. The increase of ribosomal recruitment is thought to occur as a result of the fusion partners ability to destabilize a secondary structure forming within the RBS of transcripts. Optimized distribution of ribosomes is a possible consequence of fusion partners introducing poorly adapted codons at the 5´teminal end of transcripts. This concept has not been tested experimentally previously, which makes these results especially intriguing. Collectively, the suggested mechanisms could act to increase translation rates, while stabilizing the transcript. However, these mechanisms do not explain all the trends seen in the expression data, meaning that there must be additional undiscovered mechanisms that influence expression when celB fusion partners are utilized.