6 Strategies To Increase Unstable Protein Expression In E. Coli

Article about 6 Strategies To Increase Unstable Protein Expression In E. Coli

Published by

Nov 11, 2021

For pharmaceutical target investigations, E. Coli is the most often utilized protein production system. It has significant benefits since bacterial protein synthesis is inexpensive and can produce proteins quickly. 

 

Despite its numerous benefits and extensive usage, utilizing E. coli as a translation host has certain drawbacks. Replication and translation are quick and closely connected in prokaryotic environments but not in eukaryotic ones. Because many eukaryotic proteins take longer durations and assemble chaperones to wrap into their natural form, this rate increase often results in a pool of partly folded, unstructured, or misfolded insoluble proteins. As a result, several targets, particularly more considerable multidomain and membrane molecules, fail to produce E. coli. 
 

So, can you increase unstable expression in E. coli? Yes, there are a series of steps and precautions to take while achieving the desired results. Read on to know more about creating an optimal environment for protein expression and synthesis in E. coli of highly unstable proteins. 

 

How to optimize unstable protein expression in E. coli? 

 

Once you've gathered all of the essential data and you suspect or discover difficult-to-express proteins, you may tweak several factors to improve protein expression or even prevent issues from arising during the manufacturing process. Here are some ways to Increase Unstable Protein Expression in E. coli: 


Choose the bacterial strains that are most suited: 

 

Several solutions with unique processes have been proposed in the previous decades to improve the number and quality of proteins generated in E. Coli. Various tools with different microbiological strains with special features for creating hazardous or membrane proteins, proteins with uncommon codons, or proteins with disulfide bonds are available on the market. 

 

For recombinant protein production, the strain or genetic heritage of the host strain is critical. Expression variants should be free of dangerous proteases, but they should preserve the transcription plasmid and pass the necessary genetic information to the expression vector. 

Change in media: 

 

Recombinant protein production needs nutrients for bacterial growth, and growth factors are complex to manage. This process often results in variations in resource depletion, pH, dissolved oxygen content, and the buildup of inhibitory chemicals from diverse metabolic pathways. These modifications are detrimental to the generation of soluble or properly folded active protein.  
 

Many cofactors in the culture medium, like metal ions, may be required for proper and effective protein folding. Adding these crucial components to the culture medium might significantly boost the yield of soluble proteins and their folding rate. 

 

Lower temperature expression: 
 

Protein expression in low-temperature E. coli improves the stability of proteins that are hard to produce as soluble proteins. Because hydrophobic interactions that determine inclusion body creation are temperature sensitive, expression at low temperatures leads to increased stability and proper folding patterns.  

 

Furthermore, any expression linked with a toxic phenotype exhibited at 37°C is repressed at lower temperatures. In E. coli, increasing the expression of chaperones is linked to increased expression and efficiency of lower thermal growth. As a result, growing at temperatures between 15 and 23 degrees Celsius may considerably decrease expressed protein degradation. 

Molecular chaperone co-expression: 

 

Molecular chaperones are proteins that help de novo protein function and support the correct shape of expressed polypeptides. The co-expression of the molecular chaperones method was used to inhibit inclusion body formation, resulting in improved recombinant protein solubility.  
 

Chaperones operate as a trigger factor, helping in the refolding of recombinant proteins. Even after being released from the protein-chaperone complex, these polypeptides continue to fold into their original form. Additionally, certain chaperons may inhibit protein aggregation. 

 

 

Resolubilization of E. coli integration proteins: 

 

For the commercial production of therapeutic proteins, recombinant proteins produced as inclusion structures in E. coli have been extensively exploited. Reducing recovery is one of the main downsides of refolding inclusion body proteins into a more efficient, soluble, and correctly folded product.  
 

Aside from that, the necessity for optimizing refolding parameters for each target molecule and the resolubilization technique may impact the refolded protein's function. As a result, soluble recombinant protein manufacturing remains a better option than in vitro refolding techniques. 

Inclusion body isolation: 
 

In promoting cell disintegration, lysozyme treatment combined with EDTA isolates inclusion bodies before cell homogenization. The low-speed spinning of microbes that are physically ruptured by ultrasonication homogenization recovers inclusion bodies. As inclusion body contaminants, bacterial cell wrap or extracellular matrix components will co-precipitate with the intractable fractions. 

 

Adding detergents like Triton X-100 or modest quantities of chaotropic chemicals will quickly remove these pollutants. Inclusion bodies are solubilized using varying quantities of chaotropic chemicals like urea after removing the impurities. Because of its superior chaotropic characteristics, the latter is beneficial.