A Study on Protein Expression via Sequence Reversed DNA: Investigating Activity

Abstract

Proteins expressed in recombinant cells often encounter challenges such as reduced activity or insolubility. In addressing these issues, our research focused on employing reverse transcription of DNA as a strategy. Specifically, we aimed to investigate whether proteins produced from these reconstructed sequences would retain structural and functional similarities to the target proteins or exhibit any alterations. In this study, we used chitosanase (CSN) and its signal sequence (CSP) derived from Bacillus for experimentation. We verified that CSN derived from Bacillus subtillis CH2 functions correctly when expressed in E. coli. Following the successful confirmation of CSN expression in recombinant cells, we systematically reconfigured both the CSN and CSP sequence in various ways to assess their impact on protein expression. The results of the experiment showed that retro chitosanase signal peptide (rCSP) and retro chitosanase (rCSN) was expressed as insoluble protein. This insolubility was attributed to the misfolding of these proteins, as predicted. In the experimental group where only, the CSN and connected the rCSP, protein expression resulted in solubility similar to that observed in the original recombinant protein. This result suggests that the proper CSN sequence compensates for the reversed signal peptide, allowing for appropriate folding and solubility. In summary, our study focused on investigating protein expression patterns through the manipulation of protein sequences in reverse. Furthermore, we examined the distinctions between these proteins based on the outcomes observed during the experiments.