Terri E. O'Neil

RECOMBINANT CHICKEN IFN-GAMMA EXPRESSED IN ESCHERICHIA COLI. ANALYSIS OF C-TERMINAL TRUNCATION AND EFFECT ON BIOLOGIC ACTIVITY

Interferon-gamma (IFN-gamma) possesses potent immunostimulatory properties, and it has recently been shown to have potential therapeutic properties. Recombinant protein technology is frequently used for commercial production of therapeutics, such as IFN. Biologically active recombinant chicken IFN-gamma (rChIFN-gamma) constructs bearing an N-terminal poly-His tag were expressed in Escherichia coli. Preparations of rChIFN-gamma contained varying ratios of a full-length and a truncated protein species (18 and 16 kDa, respectively). Amino acid sequence analysis of the full-length protein corroborated the sequence previously predicted from the cDNA sequence. Full-length rChIFN-gamma contains two cysteine residues at the C-terminus, and these were labeled by reduction and subsequent specific alkylation with fluorescent tag (5-I-AEDANS) to distinguish between full-length and C-terminally truncated forms of rChIFN-gamma. Comparative peptide mapping, amino acid sequencing, and mass spectrometry revealed that the 16 kDa protein was truncated at Lys133. It was also observed that the 18 kDa rChIFN-gamma protein was infrequently contaminated with small quantities of protein truncated at Arg141. A truncated recombinant construct (His1-Lys133) was also expressed in E. coli and had biologic activity comparable with that of the full-length construct. The 3-D structure of rChIFN-gamma was deduced by comparative modeling with bovine and human IFN-gamma crystallographic structures. Analysis of sequences and comparison of structures have revealed that the 3-D structure of rChIFN-gamma is similar to those of bovine and human molecules despite an overall amino acid identity of only 32%.

Characterisation of the porcine cytokines which activate the CD131βc common sub-unit, for potential immune-augmentation

Graphical abstract Recombinant cytokines IL‐3, IL‐5 and GM‐CSF induce significant increases in peripheral basophils and eosinophils in pigs. Figure. No Caption available. HighlightsrPoIL‐5 was identified as a potent eosinophilopoietin in pigs.rPoIL‐3 induces a significant increase in basophil numbers.rPoGM‐CSF significantly increases progenitors and granulocytes the BM.rPoGM‐CSF significantly increases peripheral eosinophils. Abstract Early acting cytokines and growth factors such as those of the CD131 &bgr;c subunit, may offer an alternative method to the current use of antibiotics and chemicals such as anthelmintics in maintaining Porcine (Po) health. Thus far, the recombinant Po (rPo) Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), rPo interleukin‐3 (IL‐3) and rPo interleukin‐5 (IL‐5) proteins have been identified and cloned and the biological activity of each cytokine has been confirmed in vitro, however, in vivo immune system regulation and hematopoietic stem cell (HSC) augmentation are regulated by numerous cytokines and cellular signals within the bone marrow (BM) niche. In order to quantify the use of recombinant cytokines in augmenting the immune response, it is necessary to determine the stages of hematopoiesis induced by each cytokine and possible areas of synergy requiring further investigation. Here we used the chemotherapeutic agent 5‐fluorouracil (5‐FU), to chemically induce a state of myelosuppression in young pigs. This allowed for the monitoring of both the autologous BM reconstitution and recombinant cytokine induced BM repopulation, precursor cell proliferation and cellular differentiation. The recombinant cytokines PoGM‐CSF, PoIL‐3 and PoIL‐5 were administered by intramuscular injections (i.m.) following confirmation of 5‐FU induced leukocytopenia. Blood and BM samples were collected and then analysed for cell composition. Statistically significant results were observed in several blood cell populations including eosinophils for animals treated with rPoIL‐5, rPoGM‐CSF and basophils for animals treated with rPoIL‐3. BM analysis of CD90+ and CD172a+ cells confirmed myelosuppression in week one with significant results observed between rPoIL‐3 and the 5‐FU control group in week two and for the rPoGM‐CSF group in week three. These results have demonstrated the effects of each of these rPo cytokines within the hematopoietic processes of the pig and may demonstrate similar outcomes in other mammalian models including human.