HomeLifestyleHealthHow To Increase Unstable Protein Expression In E.Coli Tips And Tricks

How To Increase Unstable Protein Expression In E.Coli Tips And Tricks

It can occasionally be complicated to generate and purify recombinant proteins for therapeutic development or in vitro investigations. Significant time and financial inputs are necessary to produce high yields of pure and functional recombinant proteins.   

This post aims to provide a guide for setting up ideal circumstances for expressing and synthesizing troublesome, unstable proteins of relevance as recombinant proteins in E. coli. 

The most used protein production system for drug target investigations is E. Coli. Because bacterial protein manufacturing is inexpensive and can produce proteins quickly, it has many benefits. Bioinformatic analysis is the first step in the comprehensive optimization pathway for creating problematic proteins. 

Six additional factors can be adjusted to optimize E. Coli protein expression or foresee potential manufacturing process issues once all this information has been gathered and you suspect or discover proteins that are difficult to express. 

Guidelines for Improving E. Coli Protein Expression 

Select the Bacterial Strains That Are Best Adapted 

Over the past few decades, several tactics with novel strategies have been designed to increase the quantity and quality of proteins generated by E. Coli. Numerous tools on the market with a wide range of bacterial strains have unique properties for producing membrane or hazardous proteins or proteins with uncommon codons or disulfide bonds. 

Select The Appropriate Expression Vector 

Multiple replicators, cloning sites, promoters, selection markers, and fusion proteins are included in the expression vectors. There are an enormous variety of plasmids on the market. Therefore, choosing the best one to produce recombinant proteins is difficult. 

There are various promoters, including: 

  • The T7 promoter 
  • The pL promoter 
  • The lac promoter 
  • The pL promoter 

Codon optimization 

The bulk of amino acids is encoded by several codons, which indicates that each amino acid is associated with multiple tRNAs. Some redundant tRNAs that code for identical amino acids are significantly more prevalent in a particular cell than others. Codon optimization is adjusting the transgene’s codons without altering the sequence of amino acids it encodes for.  

This typically boosts the protein’s availability by exchanging rare codons with plentiful codons found in the host organism.

Taking advantage of the natural ratios of tRNAs present in heterologous expression systems is generally considered to be a means of increasing the rate at which the target gene is translated.

Reduce Expression Temperature 

The use of bacterial cultivation at low temperatures to lessen protein aggregation is well recognized and well-documented. It lessens the hydrophobic interactions necessary for protein self-aggregation by slowing the pace of protein synthesis and folding kinetics. Due to the weak activity of heat shock proteases, which are often increased upon protein overproduction in E. coli, low cultivation temperatures can also limit or impede protein breakdown.  

However, this tactic has some disadvantages, as lowering the temperature can also adversely affect bacterial growth and protein yields and transcription, translation, and replication rates. However, using cold-inducible promoters, which maximize protein production in low-temperature environments, can reach these limits.  

Create Protein in Specialized Media and Adapted Environment 

Enhancing the expression of recombinant proteins can also be done at the level of culture media composition and associated chemicals. The numerous experimental setups shown in figure 2 were carried out at our R&D facilities at Tebu-corporate Bio’s office (Le Perray-en-Yvelines, France).   

These optimizations’ primary goal was to increase protein X’s solubility of protein X.

 In order to determine the optimal culture composition and conditions for viral protein X expression with the MBP tag, those studies were designed. The experiments were done on a 62 kDa MBP-fused viral protein with two disulfide bonds and a highly hydrophobic core that is extremely unstable.  

Additional Stabilizing Sequences 

Several tags can also be utilized to increase the effectiveness of protein purification and solubility. 

1. Big Tags for Stabilizing and Solubilizing 

The N- or C-terminal regions of the protein can both receive the fusion tag. The earliest tags, Protein A (280 amino acids) and LacZ were created initially to improve the protein’s solubility (1024 amino acids). 

2. Small Peptides are constituted by a single amino acid.  

Poly-amino-acid peptides, peptide sequences made up of just one type of amino acid, can be helpful in partially resolving the issue of protein solubility.

When amino acids are introduced to proteins at the C-terminus, these sequences enhance the amino acids’ adhesion, aggregation, polymerization, and solubility properties.  

Co-expressing Molecular Chaperones 

The lack of post-translational modifications (also known as PTMs), which are crucial for the lifespan and functionality of proteins, is the main drawback of the E. Coli protein expression method. 

Identifying the interaction sequences of the target protein’s partners can also significantly affect protein structure, solubility, and activity.


To sum up, every protein is a unique case. It is difficult to express an unstable protein in E. coli systems, and there are no “magic recipes.” However, solutions are available by considering their unique physico-chemistry, executing the practical design of experiments (DoE), employing new tactics, and using cutting-edge reagents. It is essential to interpret all the data and screen the outcomes carefully to accomplish this. 


Please enter your comment!
Please enter your name here

Must Read