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How To Increase Protein Expression in E.coli


How To Increase Protein Expression in E.coli

Recombinant protein expression services use a range of systems and methods to curate custom and accurate results. After choosing E. coli for expression, many look to increase the yield while maintaining the correct folding of the target proteins. Below, we explore how to increase protein expression in E. coli and the steps our lab may take to achieve these results.

Codon Optimization

Codon optimization is a process that modifies the DNA sequence of a gene of interest without altering the amino acid sequences to improve its expression in a heterologous host organism. The codons, also known as the nucleotide triplets, encode the amino acids in a protein. Codon optimization takes into account the codon usage bias of the host organism, which is the preference for certain synonymous codons over others.

Codon optimization can increase the availability of the corresponding tRNAs, which are the molecules that deliver amino acids to the ribosome during protein synthesis. This can enhance the efficiency and accuracy of translation and thus increase the yield and quality of the recombinant protein.

Induction Conditions

“E. coli induction conditions” is a much broader term for a range of characteristics, including inducer concentration, culture media, post-induction temperatures, and times. Each of these factors plays a significant role in the induction condition and the overall expression of E. coli proteins. Each parameter can be optimized separately or in combination to achieve the desired level of protein expression and solubility. Another vital factor to consider is the lysis buffer composition, as this helps increase solubility in recombinant proteins. Labs can omit detergents if necessary because some may interfere with purification methods.

Fusion Tags

Fusion tags significantly affect E. coli production yields, folding, and solubility and lend a hand in purification measures. Furthermore, if there is a case of insolubility, fusion tags can solve this issue up to 60 percent of the time.

Fusion tags are external peptides or proteins that are fused to your protein of interest. A range of tags is available, but common fusion tags used in E. coli expressions include SUMO, GST, and TRX. A SUMO tag facilitates an 80-percent expression level and improves solubility by 70 percent. The removed tag is UIP1. The GST fusion tag facilitates an expression level of 75 percent with improved solubility of 65 percent. Thrombin is the removed tag. TRX facilitates an expression level of 90 percent with 60 percent improved solubility. The removed tag is Enterokinase.

Aside from choosing the most suitable fusion tag, one must carefully consider the linker sequence. This sequence joins the proteins with the tag and is a fundamental building block to proper folding and protein functionality. Each linker sequence features its own distinct characteristics and varies in structure. Some are flexible, and others are rigid. Choosing the wrong fusion tag or linker sequence can degrade the overall functionality of the protein of interest.

Culture Condition Optimization

Optimizing culture conditions is another way to increase protein expression in E. coli. Carefully selecting the culture media and temperature can make a difference in yields and purification. Using a fermenter instead of a shake flask can also provide better control over the culture’s conditions.

Optimizing the culture conditions can increase the expression efficiency and the quality of yields. Ideal culture growth takes place at an aerobic temperature of 37 degrees Celsius. In rare cases, some E. coli strains have shown growth around 53 degrees Celsius. Still, it’s worth noting this is not typical or expected for average laboratory purposes and functions. E. coli can also survive at low temperatures, such as 4 degrees Celsius, which is commonly used for storage, but the growth rate will be significantly reduced.

The pH level of the culture medium also affects the growth and expression of E. coli. The optimal pH range for E. coli is between 6 and 8, with a neutral pH of 7 being the most common. The doubling time of E. coli under optimal conditions in rich media is about 20 minutes, which means that the cell number can increase exponentially in a short time.

Chaperone Co-Expression

Chaperones are the core of quality control for proteins. Chaperones also help polypeptides achieve their intended final structure. When working with E. coli recombinant protein expression, the lack of post-translational modifications can hinder the life span and functionality of the protein of interest. When introducing a chaperone for co-expression, the success rate increases and PTM issues could be resolved.

In addition to a PTM resolution, protein folding can benefit greatly from chaperone co-expression when the binding partner is properly matched. Chaperoning has a direct and clear response to the solubilization, conformation, and overall activity of the protein of interest.

E. coli Expression Workflow for Increased Production

Having a thorough and transparent workflow in place can help increase expression production with high-quality, reputable yields. Bon Opus follows a four-step general workflow that includes synthesizing the genes, cloning and transformation, an expression test, and purification or scaling. Throughout each step, you can expect our team to communicate successes and flaws, and we collaborate with your team to find a resolution.

Our general workflow features many smaller steps that help produce the expressed E. coli proteins with bioactivity. During gene synthesis, codon optimization will improve the host’s expression. After optimizing the codons, selecting an E. coli strain for ideal expression is vital. We proudly carry a wide range of strains.

We introduce expression vectors to the culture conditions to further drive the expression and increase production. We make careful considerations for the culture conditions and adhere to the intended temperatures and time per the selected media. We then select a fusion tag based on our professional evaluation and culture conditions, and we fuse it to the protein. Refolding occurs, and in some cases, co-expression with chaperones is necessary. It’s worth noting that co-expression can increase production yields but does not suit every E. coli expression case.

Bon Opus Biosciences offers comprehensive and affordable E. coli expression services with a thorough workflow and reliable turnaround time. Our in-house team can evaluate your host cells and communicate collaboratively about changes as they arise, ensuring you’re satisfied with the yields. Partner with our team today for your custom expression service needs.


How To Increase Protein Expression in E.coli

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