Expression and Purification of Cytokine Receptor Homology Domain of Human Granulocyte-Colony-Stimulating Factor Receptor Fusion Protein in Escherichia coli

2001 ◽  
Vol 21 (1) ◽  
pp. 87-91 ◽  
Author(s):  
Daisuke Tatsuda ◽  
Haruhiko Arimura ◽  
Hiroko Tokunaga ◽  
Matsujiro Ishibashi ◽  
Tsutomu Arakawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Cytokine Receptor ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Expression And Purification ◽  
Stimulating Factor

scholarly journals Overproduction and Purification of Soluble Recombinant Human Granulocyte Colony Stimulating Factor in Escherichia coli Using Thioredoxin as Fusion

2017 ◽  
Vol 21 (1) ◽  
pp. 1
Author(s):  
Dian Fitria Agustiyanti ◽  
Debbie Sofie Retnoningrum ◽  
Heni Rachmawati ◽  
Asrul Muhamad Fuad
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Soluble Form ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Stimulating Factor

Recombinant human Granulocyte Colony Stimulating Factor (G-CSF) has been produced in a soluble form in Escherichia coli BL21 (DE3) as a fusion protein. The open reading frame of G-CSF was synthetically constructed in previous work and was codon optimized for best expression in E. coli. In this research, the gene was fused to thioredoxin (Trx) at the N-terminal in pET32 vector. The purpose of this research was to optimize the overproduction and purification processes to obtain high yield recombinant protein in soluble form, and to characterize the Trx-G-CSF fusion protein. Overproduction was performed using IPTG induction method for 3 and 6 hours. The protein was purified by Ni-NTA affinity chromatography and separated using gradient concentration of imidazole. The purified protein was then characterized by SDS-PAGE and Western Blot analysis. Further, enterokinase was used to separate G-CSF from the fusion protein. The purified form of G-CSF was subsequently characterized using Western Blot and mass spectrometry using MALDI-TOF. The results showed that the fusion protein was successfully produced in soluble part as much as 48.25% were obtained after 3 hours of induction. The yield of  fusion protein was 67.37%  from total protein (229.65  mg protein/L culture). The Western Blot analysis showed the G-CSF band at around 18.6 kDa. Mass spectrometry with MALDI-TOF/ TOF revealed that 25.86% of amino acid residue was recognized as part of human G-CSF sequence. 

Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli

2020 ◽  
Vol 14 (4) ◽  
pp. 269-282
Author(s):  
Sadra S. Tehrani ◽  
Golnaz Goodarzi ◽  
Mohsen Naghizadeh ◽  
Seyyed H. Khatami ◽  
Ahmad Movahedpour ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Clinical Aspects ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
E Coli ◽  
Secretory Production ◽  
Stimulating Factor

Background: Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. Objective: In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. Methods: Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. Results: LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. Conclusion: Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.

The effect of rare codons following the ATG start codon on expression of human granulocyte-colony stimulating factor in Escherichia coli

2015 ◽  
Vol 114 ◽  
pp. 108-114 ◽  
Author(s):  
Zeinab Karimi ◽  
Navid Nezafat ◽  
Manica Negahdaripour ◽  
Aydin Berenjian ◽  
Shiva Hemmati ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Start Codon ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Rare Codons ◽  
Stimulating Factor

Signaling by the Granulocyte-Colony Stimulating Factor Receptor Involves Jak2-Dependent Phosphorylation of Gab2.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2173-2173
Author(s):  
Lin Wang ◽  
Jia Xue ◽  
Seth J. Corey ◽  
Lisa J. Robinson
Keyword(s):  
Fusion Protein ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Erk Phosphorylation ◽  
Stimulating Factor

Abstract Granulocyte colony stimulating factor (G-CSF) is the major cytokine involved in neutrophil production. G-CSF has pleiotropic effects on myeloid cells, initially stimulating proliferation but later promoting differentiation. The specific signaling pathways that mediate the diverse effects of G-CSF remain incompletely understood. Recently, the scaffolding molecule Grb2-associated binder protein 2 (Gab2) was shown to play an important role in G-CSF induced myeloid differentiation (Zhu et al. Blood 2004). Ligand stimulation of the G-CSF receptor results in the rapid phosphorylation of Gab2, but the identity of the responsible kinases and the molecular events dependent on Gab2 phosphorylation remain unclear. Because Janus kinases (Jaks) play a central role in G-CSF signaling, we investigated the involvement of Jaks in G-CSF-stimulated Gab2 phosphorylation using the hematologic DT40 cell line stably transduced with the human G-CSF receptor (DT40GR). Antisense Jak1 and Jak2 constructs expressed in DT40GR cells each produced a marked reduction in their target Jak protein, but only antisense Jak2 reduced G-CSF-stimulated Gab2 phosphorylation. To determine whether Gab2 phosphorylation required Jak2 kinase activity, dominant negative Jak2 mutants lacking catalytic activity were expressed in the DT40GR cells. Expression of dominant negative Jak2 inhibited Gab2 phosphorylation in response to G-CSF. Similarly, treatment with the Jak2-selective kinase inhibitor AG490 markedly reduced G-CSF-dependent Gab2 phosphorylation. Co-immunoprecipitation studies further demonstrated a G-CSF- and Gab2 phosphorylation-dependent association of Jak2 with Gab2 in vivo, which was detectable by 30 seconds after G-CSF stimulation. To determine whether Gab2 was a direct substrate of Jak2, we performed in vitro phosphorylation studies using Gab2-GST fusion protein substrates. Jak2 immunoprecipitated from G-CSF-stimulated cells, but not from control cells, phosphorylated the Gab2 fusion protein. To identify potential Jak2 tyrosine phosphorylation sites in Gab2, we used site-directed mutagenesis to produce three Gab2 tyrosine mutants. Tyrosines 409, 452, and 476 were each replaced by phenylalanine (Y409F, Y452F, and Y476F). The Y452F and Y476F mutations of Gab2 each inhibited G-CSF-stimulated Jak2-dependent phosphorylation of Gab2, both in stably-transfected DT40GR cells and in transiently-transfected 293 cells also transduced with the G-CSF receptor. In contrast, G-CSF-stimulated Gab2 phosphorylation appeared unaffected by the Y409F mutation. We also evaluated downstream events in G-CSF signaling in cells expressing these Gab2 tyrosine- mutants. Akt and Erk phosphorylation following G-CSF stimulation was inhibited by both the Y452F and Y476F Gab2 mutations, but was unaffected by the Y409F mutation. These results suggest that Jak2 may mediate G-CSF differentiation signals through Stat-independent mechanisms.

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