Construction of Recombinant Human GM-CSF and GM-CSF-ApoA-I Fusion Protein and Evaluation of Their Biological Activity

Abstract

In this study, two strains of the yeast P. pastoris were constructed, one of which produced authentic recombinant human granulocyte-macrophage colony-stimulating factor (ryGM-CSF), and the other was a chimera consisting of ryGM-CSF genetically fused with mature human apolipoprotein A-I (ApoA-I) (ryGM-CSF-ApoA-I). Both forms of the cytokine were secreted into the culture medium. The proteins’ yield during cultivation in flasks was 100 and 60 mg/L for ryGM-CSF and ryGM-CSF-ApoA-I, respectively. Both forms of recombinant GM-CSF stimulated the proliferation of human TF-1 erythroleukemia cells; however, the amount of chimera required was 10-fold that of authentic GM-CSF to induce a similar proliferative effect. RyGM-CSF exhibited a 2-fold proliferative effect on BFU-E (burst-forming units—erythroid) at a concentration 1.7 fold less than non-glycosylated E. coli-derived GM-CSF. The chimera together with authentic ryGM-CSF increased the number of both erythroid precursors and BMC granulocytes after 48 h of incubation of human bone marrow cells (BMCs). In addition, the chimeric form of ryGM-CSF was more effective at increasing the viability of the total amount of BMCs, decreasing apoptosis compared to the authentic form. ryGM-CSF-ApoA-I normalized the proliferation, maturation, and segmentation of neutrophils within the physiological norm, preserving the pool of blast cells under conditions of impaired granulopoiesis. The chimera form of GM-CSF exhibited the properties of a multilinear growth factor, modulating the activity of GM-CSF and, perhaps, it may be more suitable for the normalization of granulopoiesis.

Engineering a New IFN-ApoA-I Fusion Protein with Low Toxicity and Prolonged Action

“…This study is not the first time that we have been faced with a low level of synthesis of ApoA-I and chimeric proteins containing ApoA-I [46]. Although genes for chimeric proteins were engineered and optimized for expression in yeast, the yield of the secreted chimeric proteins varied significantly [47,48]. The yield of proteins could most likely be significantly increased by carrying out cultivation in a bioreactor.…”

“…It is noteworthy that the addition of Tween 20 to the culture medium in the induction phase allowed a significant increase in the yield of both ryIFN and its chimera (by approximately a factor of three). The addition of Tween 20 has had a positive effect on the production yields of other recombinant yeast proteins obtained by us (G-CSF and G-CSF-ApoA-I [48,49]; GM-CSF and GM-CSF-ApoA-I [47]). There are a lot of data on the effect of non-ionic detergents not only increasing the secretion of target proteins but also increasing the stability of the protein in solution and preventing its aggregation [50,51].…”

Evaluation of the biological activity of the GM-CSF-ApoA-I fusion protein, obtained by biosynthesis in the yeast <em>Pichia pastoris</em>

“…RyGM-CSF-ApoA-I stimulated granulopoiesis comparable to authentic cytokine. RyGM-CSF-ApoA-I maintained proliferation, decreased apoptotic cell death, and retained the pool of granulocyte progenitor BMCs more efficiently than ryGM-CSF 5 .…”

Eukaryotic Expression of the Cytochrome c Oxidase Subunit I of Sitophilus zeamais and Its Interaction with Allyl Isothiocyanate

“…The strains were sequenced to identify positive strains using specific forward primers and universal reverse primers. In order to screen multicopy genes, the transformants were inoculated on medium containing a higher concentration of zeocin (500, 1000, and 2000 μg/mL) and incubated at 30 °C for 2–4 days until single colonies of the transformants were formed. , …”

“…In order to screen multicopy genes, the transformants were inoculated on medium containing a higher concentration of zeocin (500, 1000, and 2000 μg/ mL) and incubated at 30 °C for 2−4 days until single colonies of the transformants were formed. 26,27 Expression of the Recombinant COX I Protein in P. pastoris. Single colonies of transformants grown on agar plates containing higher concentrations of zeocin were inoculated into 5 mL of buffered glycerol complex medium (BMGY; 1% [w/v] yeast extract, 2% [w/v] peptone, 100 mM KH 2 PO 4 buffer [pH 6.0], 1.34% [w/v] yeast nitrogen base, 4 × 10 −5 % [w/v] biotin, and 1% [v/v] glycerol) and shaken at 30 °C and 250 rpm for 16−18 h. The next day, 250 μL of culture was transferred to 25 mL of BMGY medium and incubated at 30 °C for 16−18 h at 250 rpm until the OD 600 reached 1−2.…”