IL1A is a critical pro-inflammatory cytokine in the immune system. Inflammatory response is played out and controlled through this. IL1A is produced by activated macrophages and epithelial cells and other cell types and mediates a variety of immune and inflammatory responses.
Due to the biotechnological advancement, recombinant IL1A can be produced with important research and therapeutic applications.
The aim of this article is to describe the process of human recombinant IL1A production in E.coli bacteria, as well as to analyze its molecular structure and the methods of its production and purification.
Structure of Recombinant IL1A
This is recombinant IL1A produced in E. coli as a single, non glycosylated polypeptide chain of 297 amino acids (113–271 a.a.). Its molecular mass is about 22.4 kDa. To increase stability and facilitate purification, the protein is fused at its N terminus with a 38 amino acid His-tag.
This is a widely used affinity tag in recombinant protein production including this His tag and the protein could be isolated using nickel based chromatographic methods effectively.
Proteins expressed in E. coli lack post-translational modification leading to the lack of glycosylation. Although this limitation exists, the recombinant IL1A is functional and structurally intact and hence is a useful tool for scientific investigations.
| Gene Cloning → Transformation into E. coli → Induction of Expression → Cell Lysis → Protein Purification → Elution → Quality Testing → Final Product |
Production of Recombinant IL1A in E. coli
Why E. coli?
E. coli is selected as a host organism for IL1A production because of its simplicity, rapid growth and inexpensive nature. While production of recombinant proteins is well established in E. coli systems, these systems yield high levels of production in a fairly short time frame. Moreover, they are readily manipulatable genetically, which makes them perfect for large scale protein production.
Expression System
In plasmid based expression systems recombinant IL1A is usually expressed by E. coli. An expression vector is then obtained which encodes the IL1A protein including the N-terminal His tag. E. coli cells will then be transformed with this vector. Recombinant IL1A protein is expressed in cytoplasm upon the induction with agents such as isopropyl β- D- 1- thiogalactopyranoside (IPTG).
Challenges in Expression
E. coli provides advantages, but challenges such as the formation of inclusion bodies exist with it. These consist of aggregated misfolded proteins that must be solubilized and refolded to regain biological activity. There are some means to alleviate the inclusion of body formation, including the optimization of growth conditions, i.e. temperature, pH and induction timing.
Purification of Recombinant IL1A
Proprietary chromatographic techniques used for purification of recombinant IL1A provides high purity and biological activity. The N-terminus His tag affords more simple purification; this enables affinity chromatography with nickel or cobalt resin.
5 Easy Steps in Purification
1. Cell Lysis
Recombinant IL1A is expressed in E. coli cells and are harvested to subsequently lyse the cells to release intracellular proteins. Mechanical method (e.g., sonication) or enzymatic method (e.g., lysozyme treatment) can achieve the lysis.
2. Affinity Chromatography
The His-tag will bind to columns immobilized with metal affinity chromatography (IMAC). The His-tagged protein can then be bound to nickel or cobalt ions in the column, meaning all other impurities will be washed away.
3. Elution
For elution of bound IL1A, an imidazole containing buffer is used to complete the His-tag for binding to the resin. In this step IL1A protein is purified to a very high level.
4. Refolding (if necessary)
If an expression in inclusion bodies is needed, the protein must be solubilized from them using denaturants such as urea or guanidine hydrochloride. Secondly, the protein is refolded by removal, in a step‑wise manner, of the denaturant with or without the introduction of stabilizing agents.
5. Quality Control
IL1A purified by us is subject to rigorous quality control for purity, identity and activity. The quality of the product is confirmed using techniques, such as SDS-PAGE, Western blotting, and bioassays.
4 Applications of Recombinant IL1A
1. Research
Recombinant IL1A is extensively utilized for immunological research to investigate the inflammation and immune response. As a tool to recruit and activate varied signaling networks, it is a key tool for the investigation of cytokine networks and cellular interactions.
2. Drug Development
IL1A is used in drug screening assays in the field of pharmaceutical industry to discover anti-inflammatory or immunomodulatory compounds. IL1A reported here serves as a model cytokine to test inhibitors targeting IL1A signaling pathways.
3. Disease Models
IL1A is used in experimental models to mimic inflammatory conditions, about which therapeutic interventions can be developed in rheumatoid arthritis, psoriasis and inflammatory bowel disease.
4. Biotechnology and Diagnostics
The characterization and production of IL1A can also be used to develop diagnostic tools to detect inflammatory markers in clinical samples.
Advantages of Recombinant IL1A Production in E. coli
- Cost-Efficiency: E. coli cultivation is cheap and easily scalable for large scale production.
- Scalability: This system is easily scalable to large production amounts for industrial applications.
- High Yield: High yields of target proteins are obtained by optimized expression systems in E. coli.
- Ease of Purification: His tag makes purification more convenient and less expensive.
Limitations and Future Directions
Although E. coli derived IL1A has benefits, it is not glycosylated and this could affect its activity in certain applications. Alternative expression systems, for example in yeast or mammalian cells, may serve as a future target of research to produce glycosylated IL1A.
Refolding techniques to improve the quality and yield of recombinant IL1A will also benefit from improved separation methods.
Final Words
Recombinant IL1A expressed in E. coli is a precious source for various scientific and industrial applications. With a His-tag fusion, it is efficiently produced and purified and its non glycosylated structure is essential to study inflammation and immune responses.
The development of recombinant proteins, such as IL1A, will only become more refined as biotechnological advancements continue, creating new opportunities for research and therapeutics.
