You have cloned a gene for an enzyme that degrades lipids in a bacterium that normally lives in cold temperatures. You wish to transfer this gene into E. coli to produce industrial amounts of enzyme for use in laundry detergent. You have managed to produce transgenic E. coli expressing mRNA of your gene, but only a low level of protein is produced. Why might this be so? How could you overcome this problem?

Key Concepts

Gene Expression Regulation

Gene expression regulation refers to the mechanisms that control the timing and amount of gene product (protein) produced in a cell. In prokaryotes like E. coli, factors such as promoter strength, ribosome binding sites, and transcription factors can significantly influence the level of mRNA and, consequently, protein synthesis. If the cloned gene's regulatory elements are not optimal for E. coli, it may lead to low protein production.

Codon Optimization

Codon optimization involves modifying the DNA sequence of a gene to use codons that are more frequently used by the host organism, in this case, E. coli. Different organisms have varying preferences for specific codons, and using less common codons can result in inefficient translation and low protein yield. By optimizing the gene sequence for E. coli's codon usage, one can enhance protein expression levels.

Post-Translational Modifications

Post-translational modifications (PTMs) are chemical changes that occur to a protein after its synthesis, affecting its activity, stability, and localization. E. coli may not perform the same PTMs as the original organism from which the gene was cloned, potentially leading to an inactive or unstable enzyme. Understanding and engineering the necessary PTMs can help improve the functionality and yield of the expressed protein.