Certain proteins are challenging to express due to a combination of intrinsic properties of the protein and limitations of the expression system. The main reasons include: 

1.   Protein Instability: 

     ●      Description: Some proteins are inherently unstable, prone to misfolding or degradation during or after synthesis. 

     ●      Impact: This leads to low yields as the protein may degrade rapidly, misfold, or aggregate, especially in heterologous systems. 

2.Toxicity to Host Cells: 

     ●      Description: Proteins that are toxic to the host cells can interfere with normal cellular processes, leading to cell death or poor cell growth. 

     ●      Impact: This reduces the overall yield, as the expression of toxic proteins can stress or kill the host cells before sufficient protein is produced. 

3.Incorrect Folding: 

     ●      Description: Some proteins, especially complex eukaryotic or membrane proteins, require specific chaperones or cofactors for proper folding, which may not be present in the expression system (e.g., bacterial systems). 

     ●      Impact: This results in the production of inactive or misfolded proteins, which may aggregate or form inclusion bodies. 

4.Post-Translational Modifications: 

     ●      Description: Many eukaryotic proteins require specific post-translational modifications (e.g., glycosylation, phosphorylation) for proper function, which are not fully supported in simpler expression systems such as bacteria. 

     ●      Impact: The lack of necessary modifications can lead to non-functional or unstable proteins. 

5.Codon Usage Bias: 

     ●      Description: Different organisms prefer different codons for the same amino acids. If the codon usage in the gene is not optimized for the host organism, it can lead to inefficient translation. 

     ●       Impact: This can slow down or block protein synthesis, reducing expression levels. 

6.Membrane Proteins: 

     ●      Description: Membrane proteins are particularly difficult to express because they require proper insertion into lipid bilayers, which may not be adequately supported by the host cell membrane environment. 

     ●      Impact: This can lead to aggregation, misfolding, or improper localization, making them hard to express in sufficient quantities. 

7.Large Size or Complex Structure: 

     ●     Description: Large proteins or those with complex quaternary structures (e.g., multi-domain or multi-subunit proteins) are more prone to misfolding and aggregation. 

     ●      Impact: These proteins are harder to express because they may require specific folding pathways or assembly mechanisms that are inefficient in the host system. 

Proteins are difficult to express due to factors such as instability, toxicity to host cells, incorrect folding, lack of necessary post-translational modifications, codon usage bias, challenges with membrane protein expression, and the complexity of large or multi-subunit proteins. These issues require careful optimization of the expression system and conditions to overcome. 

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.