Purification and Characterization of Recombinant Truncated Human Interleukin-11 Expressed as Fusion Protein in Escherichia coli

Abstract

Mature human interleukin-11 (HuIL-11) is a cytokine consisting of 178 amino acid residues that results from scission of the N-terminal signal peptide, consisting of 21 amino acid residaues, from the corresponding nascent polypeptide. A DNA fragment encoding a truncated HuIL-11 (trHuIL-11), with an additional 5 amino acid residues removed from the N-terminus, was cloned into vector pGEX-2T between the BamHI site and the EcoRI site. Upon transformation with Escherichia coli BL21, the construct over-produced a glutathione S-transferase (GST)-fused protein in a soluble form after IPTG induction. The fusion protein was initially fractionated with butyl-Sepharose 4 fast flow column and by affinity chromatography using a GSH-Sepharose 4B column. On-site enzymatic release with thrombin gave the target protein at 96% purity as judged by SDS-PAGE and HPLC. Expression of the interleukin as a GST-fused protein thus greatly improved downstream processing. Subsequent biological activity assay suggested that trHuIL-11 had similar activity profile to the naturally produced sample and may be a promising candidate for further development as biopharmaceutical.

A panoramic review and in silico analysis of IL-11 structure and function

“…The precursor protein contains 199 aas (MW=23 kDa), including a signal peptide sequence of 21 aas, which is cleaved off before releasing the protein from the cell [7]. IL-11 is very basic due to a high abundance of leucine (23%), proline (12%), and positively charged aas (14%) [4,8].…”

“…Variations in the N-terminal region up to residue 60, compared to the other parts, are more frequent. In this context, results from several investigations suggested that the bioactivity of hIL-11 is mostly dependent on its C-terminal, while in some mutational studies using alanine scanning or a truncation method, the N-terminal was identified as an unstructured non-functional region [6,7,23,27]. Because an unstructured region usually brings susceptibility to proteolysis, the N-terminal part has been targeted for removal in attempts to obtain a more stable protein against chemical and proteolytic stresses.…”

“…Because an unstructured region usually brings susceptibility to proteolysis, the N-terminal part has been targeted for removal in attempts to obtain a more stable protein against chemical and proteolytic stresses. For example, Tan et al [7] produced a truncated human IL-11 that lacked the five aas residues of the N-terminal, which indicated an almost identical biological activity compared to the fulllength rhIL-11. On the other hand, the C-terminal region plays a role in stability, as well as biological activity of IL-11, considering that the deletion of four aas from the C-terminal leads to protein insolubility and a significant decrease in biological activity [19].…”

“…IL-11 is produced by many cells with mesenchymal origin, including keratinocytes, synoviocytes, chondrocyte, osteoblasts, leukocytes, fibroblasts, and epithelial cells [7,11].…”

Non-core Region Modulates Interleukin-11 Signaling Activity

“…8. Interleukin 11, contrary to other cytokines such as hIL-6 or granulocyte colony-stimulating factor (45,46), is not glycosylated in its native form (47). Dissimilar glycosylation patterns among interleukins could contribute to the fascinating diversity of signaling events elicited by this structurally homologous family of cytokines.…”