How Recombinant Proteins are Expressed in Escherichia coli
The successful expression of recombinant proteins in Escherichia coli (E. coli) is one of the central advancements in biosciences.
E. coli has become a standard host in biotechnology because of its easy genetic tractability, rapid growth rate, and cost-effectiveness. Used for the production of a diverse array of proteins, enzymes, hormones, and pharmaceutical-grade antibodies, this capability of the microorganism has fueled academic study.
We will understand how rProteins are expressed in E.coli and what makes this tech an essential part of modern research.
Mechanism of Recombinant Protein Expression
Before we understand the inside-out mechanism of recombinant protein expression, we need to understand what recombinant proteins are.
At the core, recombinant proteins are artificially produced proteins that are produced by introducing foreign genes into an organism. The gene encoding the target protein is inserted into E.coli, and the cellular mechanism of the bacteria synthesizes it in a usable and measurable quantity.
Many of the recombinant proteins include recombinant antibodies that are a special type of rProtein mimics or enhance the immune response.
Steps Involved in E. coli Expression
In most cases, the following steps are involved in the process of expression:
01. Gene Cloning
The gene of interest is ligated into the circular piece of DNA, called a plasmid, because it contains elements such as promoters and antibiotic resistance genes, which help in protein expression.
02. Transformation
Once the recombinant plasmid is formed, it is introduced into E. coli cells through the process of heat shock or electroporation. It allows them to take up the foreign DNA.
03. Selection & Cultivation
Selective media containing antibiotics are used for growing transformed cells.
04. Induction of Protein Expression
Protein production needs to be induced after sufficient biomass is produced. This is achieved by adding a chemical like IPTG to activate the promoter that controls the inserted gene.
05. Harvesting & Purification
Cells are harvested and lysed after they are successfully expressed. Purification methods, including affinity chromatography and other techniques such as SDS-PAGE, are used for the verification of recombinant proteins.
Optimizing Strategies and Addressing Challenges in Recombinant Protein Expression
The issue of solubility and folding does occur, even though E. coli is efficient at producing recombinant proteins. Some proteins with many disulfide bonds often form inactive aggregates called inclusion bodies.
Researchers have developed strategies to address these challenges. Let’s go through them, one by one:
01. Periplasmic Targeting
This involves directing proteins to the periplasmic space. It is effective for complex proteins and certain functional antibody fragments, as the environment in the periplasmic space favors correct disulfide bonds.
02. Multiple Host Strains
Some specialized strains of E. coli, like BL21(DE3), C41(DE3), or Lemo21(DE3), help in mitigating issues of folding and toxicity by enhancing chaperone availability and tuning expression levels.
03. Chaperone Proteins and Fusion Tags
For correct folding and to increase the yield of active protein, solubility-enhancing fusion tags and co-expressing molecular chaperones are added to the selective media.
04. Codon Optimization and Growth Conditions
To ensure efficient translation and higher protein output, gene sequences are adapted to match the codon usage of E. coli better.
Additionally, alterations in growing conditions, such as adjustments in temperature, concentration, and nutrient composition, affect protein activity and solubility.
The Production of Recombinant Antibodies: E. coli Based
In modern research, recombinant antibodies are indispensable, and hence their E. coli based production is attractive because of its cost-effectiveness and scalability.
There are some issues that need to be tackled, and advances in molecular biology allow for the production of different antibody formats in E. coli. It includes Fab fragments and single-chain variable fragments (scFvs) as well.
Trusted companies such as AAA Biotech have achieved a high level of trust in the scientific community for providing highly validated recombinant antibodies for research purposes.
Final Thoughts
"Research is to see what everybody else has seen, and to think what nobody else has thought."
- Albert Szent-Györgyi
From offering versatility, efficiency, and scalability for the production of enzymes to recombinant antibodies, the expression of recombinant proteins is one of the cornerstones of biotechnology.
The ongoing work of optimizing host strains, molecular tools, and process parameters allows researchers to push the boundaries of what can be achieved in bacterial expression systems.
