Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in Bombyx mori pupae

Chen, Jian; Nie, Zhou; Zhengbing, Lu; Zhu, Chenggang; Xu, Chengzhi; Jin, Yongfeng; Wu, Xiang-Fu; Zhang, Yaozhou

doi:10.1007/s12010-007-9217-1

Cited by 6 publications

(5 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The purified rhGM-CSF expressed by the silkworm pupae bioreactor was produced in our laboratory . The human GM-CSF ELISA kits were obtained from Beijing 4A Biotech Co., Ltd. (Beijing, China).…”

Section: Methodsmentioning

confidence: 99%

“…In brief, solid ammonium sulfate was slowly added to the SLP solution to a final concentration of 25%, and the resulting mixture was stirred at 4 °C for 2 h. Next, the solution was centrifuged at 12000 g at 4 °C for 30 min. The supernatant was adjusted to 65% (NH 4 ) 2 SO 4 , and the precipitated proteins were pelleted . The resulting pellet was resuspended in an appropriate volume of PBS.…”

Section: Methodsmentioning

confidence: 99%

“…The purified rhGM-CSF expressed by the silkworm pupae bioreactor was produced in our laboratory. 9 The human GM-CSF ELISA kits were obtained from Beijing 4A Biotech Co., Ltd. (Beijing, China). The silkworm pupae were supplied by Zhejiang Chinagene Biopharmaceutical Co., Ltd. (Zhejiang, China).…”

Section: Methodsmentioning

confidence: 99%

“…The supernatant was adjusted to 65% (NH 4 ) 2 SO 4 , and the precipitated proteins were pelleted. 9 The resulting pellet was resuspended in an appropriate volume of PBS. Then proteins of various fractions from the SLP were collected according to the protocol described in Figure 1.…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Synergetic Protein Factors That Improve rhGM-CSF Absorption via an Oral Route Exist in Silkworm Pupae

et al. 2015

View full text Add to dashboard Cite

Recent studies have demonstrated that recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) produced by the silkworm pupae bioreactor is absorbed into blood through oral administration and functions as an active cytokine. The aim of this study was to further examine and identify synergetic protein factors in silkworm pupae that improve rhGM-CSF absorption via an oral route. The concentrations of rhGM-CSF in serum were evaluated in mice after oral administration of rhGM-CSF using different chemical compositions of silkworm pupae as pharmaceutical excipients. The experimental data revealed that the supernatant lyophilized powder (SLP) of a homogenized slurry of silkworm pupae caused a significant increase in the rhGM-CSF level in blood when rhGM-CSF was orally administered with SLP, suggesting that synergetic protein factors that improve the oral absorption of rhGM-CSF primarily exist in SLP. As shown by scanning electron microscopy, microspheres were formed when rhGM-CSF was coated with SLP. Animal experimental data showed that the absorption of orally administered rhGM-CSF through the gastrointestinal (GI) tract primarily resulted from protein factors present in the SLP retentate obtained after 10 kDa ultrafiltration. Surface plasmon resonance spectroscopy analysis demonstrated that several protein factors present in the SLP retentate obtained after 10 kDa ultrafiltration were bound to rhGM-CSF. Proteins bound to rhGM-CSF by liquid chromatography-mass spectrometry were identified as chymotrypsin inhibitor SCI-II precursor, cationic peptide CP8 precursor, Kazal-type proteinase inhibitor, and chymotrypsin inhibitor SCI-I. These findings indicate that these proteinase inhibitors play an important role in improving rhGM-CSF absorption in the GI tract.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Synergetic Protein Factors That Improve rhGM-CSF Absorption via an Oral Route Exist in Silkworm Pupae

et al. 2015

View full text Add to dashboard Cite

show abstract

“…It was purified and determined to have molecular weigh of 29 kDa [7], [10], [11]. It's post-translational modifications were characterized in addition to its glycosylation [7], [10], [11]., The oral formulation of BmrhGM-CSF was used in our previous animal study. The results from preclinical studies demonstrated that orally administered BmrhGM-CSF could function as an active cytokine [10].…”

Section: Introductionmentioning

confidence: 99%

Bioavailability of Orally Administered rhGM-CSF: A Single-Dose, Randomized, Open-Label, Two-Period Crossover Trial

Zhang

Nie

et al. 2009

PLoS ONE

Self Cite

View full text Add to dashboard Cite

BackgroundRecombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is usually administered by injection, and its oral administration in a clinical setting has been not yet reported. Here we demonstrate the bioavailability of orally administered rhGM-CSF in healthy volunteers. The rhGM-CSF was expressed in Bombyx mori expression system (BmrhGM-CSF).Methods and FindingsUsing a single-dose, randomized, open-label, two-period crossover clinical trial design, 19 healthy volunteers were orally administered with BmrhGM-CSF (8 µg/kg) and subcutaneously injected with rhGM-CSF (3.75 µg/kg) respectively. Serum samples were drawn at 0.0h, 0.5h ,0.75h,1.0h,1.5h,2.0h ,3.0h,4.0h,5.0h,6.0h,8.0h,10.0h and 12.0h after administrations. The hGM-CSF serum concentrations were determined by ELISA. The AUC was calculated using the trapezoid method. The relative bioavailability of BmrhGM-CSF was determined according to the AUC ratio of both orally administered and subcutaneously injected rhGM-CSF. Three volunteers were randomly selected from 15 orally administrated subjects with ELISA detectable values. Their serum samples at the 0.0h, 1.0h, 2.0h, 3.0h and 4.0h after the administrations were analyzed by Q-Trap MS/MS TOF. The different peaks were revealed by the spectrogram profile comparison of the 1.0h, 2.0h, 3.0h and 4.0h samples with that of the 0.0h sample, and further analyzed using both Enhanced Product Ion (EPI) scanning and Peptide Mass Fingerprinting Analysis. The rhGM-CSF was detected in the serum samples from 15 of 19 volunteers administrated with BmrhGM-CSF. Its bioavailability was observed at an average of 1.0%, with the highest of 3.1%. The rhGM-CSF peptide sequences in the serum samples were detected by MS analysis, and their sizes ranging from 2,039 to 7,336 Da.ConclusionsThe results demonstrated that the oral administered BmrhGM-CSF was absorbed into the blood. This study provides an approach for an oral administration of rhGM-CSF protein in clinical settings.Trial Registration www.chictr.org ChiCTR-TRC-00000107

show abstract

Molecular cloning, expression and characterization of a novel vacuolar protein sorting 4 gene in silkworm, Bombyx mori

et al. 2012

View full text Add to dashboard Cite

The vacuolar protein sorting 4 (Vps4) protein is essential for the multivesicular body (MVB) pathway, virus budding process and cytokinesis. Vps4 has been identified and characterized from many species, but not from silkworm Bombyx mori. In this study, we firstly identified and cloned the silkworm homologous gene for VPS4, expressed it in Escherichia coli, purified and characterized the protein designated as BmVps4. The BmVps4 cDNA contains an open reading frame of 1,314 bp, and encodes a protein of 438 amino acid residues. BmVps4 is of high sequence-similarity to Vps4 proteins from other species. The recombinant BmVps4 shows ATPase activity, which can be stimulated by Mg(2+) and inhibited by dominant mutations. Together, our data suggest BmVps4 is the genuine silkworm homologue of Vps4. To our knowledge, this is the first-time characterization of any silkworm MVB proteins. This study will facilitate further investigation of silkworm MVB pathway and its possible roles in the infection and budding of B. mori nuclear polyhedrosis virus (BmNPV), which is one of the most common and severe pathogens for silkworms. The cloned BmVps4 sequence is deposited in GenBank (Accession number GQ995504).

show abstract

Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in Bombyx mori pupae

Cited by 6 publications

References 25 publications

Synergetic Protein Factors That Improve rhGM-CSF Absorption via an Oral Route Exist in Silkworm Pupae

Synergetic Protein Factors That Improve rhGM-CSF Absorption via an Oral Route Exist in Silkworm Pupae

Bioavailability of Orally Administered rhGM-CSF: A Single-Dose, Randomized, Open-Label, Two-Period Crossover Trial

Molecular cloning, expression and characterization of a novel vacuolar protein sorting 4 gene in silkworm, Bombyx mori

Contact Info

Product

Resources

About