Undergraduate Research Spotlight: Producing Human IL11 Cytokine for Pulmonary Fibrosis Studies
This year, our undergraduate project focused on the creation and purification of human IL11 cytokine—a protein with significant implications for the study of Idiopathic Pulmonary Fibrosis (IPF). IL11 transmits signals through the IL11RA and IL6ST receptors, stimulating cell proliferation and contributing to fibrotic processes. Notably, the cytokine protein is found at elevated levels in patients with IPF, especially in airway epithelial cells and macrophages. Since there is currently no cure for pulmonary fibrosis, and typical life expectancy post-diagnosis is just five years, ongoing research is essential. Presently, treatments aim to slow lung scarring, relieve symptoms, and maintain the best possible quality of life through medication, oxygen therapy, rehabilitation, transplants, and lifestyle adjustments.
Our students began by synthesizing a DNA sequence (ID P20809), engineered with six histidines at the N-terminus to facilitate purification. The DNA codons were optimized for E. coli expression and then inserted into a pET vector with a T7 promoter. Using heat shock, the recombinant DNA was introduced into BL21 E. coli cells, which were subsequently plated on agar containing kanamycin. This antibiotic selection ensured that only cells carrying the desired DNA would grow. From a single colony, cultures were expanded in kanamycin-containing media.
Once the cell cultures reached optimal density, the temperature was lowered, and IPTG was added to induce IL11 protein expression. After incubation, the cells were removed by centrifugation which were then lysed with detergent to open the cells. The cell debris and vesicles are removed again by centrifugation leaving behind our target protein and other host cell protein.
To purify the IL11 cytokine, we took advantage of the six-histidine tag, which binds specifically to nickel-coated beads. Using an AKTA™ system, the supernatant was passed over these beads, allowing the cytokine to adhere while unwanted proteins were washed away with salt and low concentrations of imidazole. The desired protein was then eluted with higher imidazole levels.
Eluted fractions were analyzed using polyacrylamide gel electrophoresis, which separates proteins by size and charge. Fractions with high purity were pooled as the IL11 product. Further validation was performed with western blotting: after transferring the proteins to nitrocellulose paper, we probed with anti-histidine and anti-IL11 antibodies, visualizing the results with fluorescent tags.
The students purified IL11 protein varied in purity from 70–90% by HPLC size exclusion. The protein was pooled and further refined by preparative size exclusion chromatography in PBS solution for collaboration with other research groups.
This project provided our students with valuable, real-world experience—from designing DNA constructs to producing and characterizing a biological protein. Many have gone on to graduate studies or secured science-related jobs. Looking ahead, we’re excited to guide the next cohort deeper into the world of biomedical research!
Sources:
J. Swaney et.al. A First-in-Class IL-11 Receptor Blocking Antibody as a Treatment for Pulmonary Fibrosis. Am J Respir Crit Care Med 2023;207:A6262.
Rosa K Kortekaas et.al. Interleukin-11 disrupts alveolar epithelial progenitor function. ERJ Open Res. 2023 May; 9(3): 00679-2022.
Figure 1. Amino acid sequence (Blue) and nucleotide DNA sequence (Red) which was inserted into a pET vector.
Figure 2. Poly acrylamide gel of human IL-11 purified over nickel bead.
Figure 3. Western blot analysis of cells lysed containing the human IL-11 protein. The pellet fraction are the proteins contained in vesicles called inclusion bodies. These proteins tend to be unstructured and require additional modification before or after initial purification. The soluble fraction is the protein contained in the cytoplasm of the cell. These proteins tend to be structured and active. The product shown was taken from the soluble fraction.
