Inclusion bodies are insoluble aggregates of proteins that can form during the expression of recombinant proteins in prokaryotic expression systems, such as E. coli. The primary causes of inclusion body formation include: 

     1.     High Protein Expression Levels: Overexpression of recombinant proteins can overwhelm the protein-folding machinery of the cell. Excessive amounts of protein can aggregate into inclusion bodies when the cell cannot process or fold them properly. 

     2.     Misfolding of Proteins: Proteins may fold incorrectly or adopt non-native conformations. Misfolded proteins can aggregate into insoluble forms, leading to inclusion body formation. 

     3.     Cellular Stress: High levels of protein expression can induce stress in the host cells. Stress can disrupt the normal protein-folding process and contribute to the formation of inclusion bodies. 

     4.     Protein Characteristics: Some proteins have intrinsic properties that make them prone to aggregation. Proteins with high hydrophobicity or those that form disulfide bonds may be more likely to form inclusion bodies. 

     5.     Suboptimal Expression Conditions: Conditions such as temperature, pH, or media composition that are not ideal for protein expression can affect protein folding. Suboptimal conditions can lead to increased aggregation and inclusion body formation. 

     6.     Lack of Chaperone Proteins: Chaperone proteins help in the proper folding of recombinant proteins. Absence or insufficient levels of chaperones can result in improper folding and aggregation of proteins. 

Inclusion body formation is a common challenge in recombinant protein production, particularly in prokaryotic systems. It is caused by high expression levels, misfolding, cellular stress, the intrinsic properties of the protein, suboptimal expression conditions, and a lack of chaperone proteins. Addressing these factors through optimization of expression conditions and co-expression of chaperones can help reduce the formation of inclusion bodies. 

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.