1.Promoter Strength: The promoter controls the initiation of transcription. By selecting promoters with varying strengths, you can regulate the level of gene expression. Strong promoters like T7 can lead to high expression, but may also cause cell stress, while weaker promoters might result in more balanced expression. 

2.  Codon Usage: Different organisms have preferences for certain codons. Optimizing the gene sequence to match the codon usage bias of E. coli can improve translation efficiency and protein yield, avoiding rare codons that may slow down protein synthesis. 

3.  Induction Conditions: Adjusting factors such as the concentration of the inducer (e.g., IPTG for T7 systems), temperature, and induction timing can significantly influence protein expression levels and solubility. Lower temperatures can help produce more soluble proteins, while different IPTG concentrations can fine-tune expression levels. 

4.  Host Strain: Various E. coli strains are available with specific traits, such as protease-deficient strains (e.g., BL21) that help reduce protein degradation, or strains optimized for disulfide bond formation or for expressing toxic proteins (e.g., Rosetta, Origami). 

5.  Plasmid Copy Number: High-copy plasmids can lead to more protein production, but they also impose a metabolic burden on the host cell. Low-copy plasmids might produce less protein but allow for better cell health and stability, especially for toxic proteins. 

6.  Culture Conditions: Factors like the composition of the growth medium (rich media vs minimal media), pH, oxygen availability, and shaking speed can affect cell growth and protein production. For example, adding certain supplements (e.g., glucose, glycerol) may enhance protein yield. 

By carefully optimizing parameters such as promoter strength, codon usage, induction conditions, host strain, plasmid copy number, and culture conditions, the efficiency and quality of protein expression in an E. coli system can be significantly improved. 

GenCefe provide one-stop solutions from codon optimization, gene synthesis, to protein expression and purification. In addition to four conventional expression systems of bacterial, yeast, insect cell, and mammalian cell, we have also developed a patented cell free expression technology, which is suitable for the production of difficult proteins such as toxic proteins and membrane proteins.