scholarly journals Differential role of segments of α-mating factor secretion signal in Pichia pastoris towards granulocyte colony-stimulating factor emerging from a wild type or codon optimized copy of the gene

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Sakshi Aggarwal ◽  
Saroj Mishra
Keyword(s):  
Amino Acids ◽  
Pichia Pastoris ◽  
Fine Tuning ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Secretion Signal ◽  
Cargo Protein ◽  
Stimulating Factor

Abstract Background The methylotrophic yeast, Pichia pastoris has been widely used for the production of human therapeutics, but production of granulocyte colony-stimulating factor (G-CSF) in this yeast is low.The work reported here aimed to improve the extracellular production of G-CSF by introducing mutations in the leader sequence and using a codon optimized copy of G-CSF. Bioinformatic analysis was carried out to propose an explanation for observed effect of mutations on extracellular G-CSF production. Results Mutations in the pro-region of the α-mating type (MAT) secretory signal, when placed next to a codon optimized (CO)-GCSF copy, specifically, the Δ57–70 type, led to highest G-CSF titre of 39.4 ± 1.4 mg/L. The enhanced effect of this deletion was also observed when it preceded the WT copy of the gene. Deletion of the 30–43 amino acids in the pro-peptide, fused with the wild type (WT)-GCSF copy, completely diminished G-CSF secretion, while no effect was observed when this deletion was in front of the CO-GCSF construct. Also, Matα:Δ47–49 deletion preceding the WT-GCSF dampened the secretion of this protein, while no effect was seen when this deletion preceded the CO-GCSF copy of the gene. This indicated that faster rates of translation (as achieved through codon optimization) could overcome the control exercised by these segments. The loss of secretion occurring due to Δ30–43 in the WT-GCSF was partially restored (by 60%) when the Δ57–70 was added. The effect of Δ47–49 segment in the WT-GCSF could also be partially restored (by 60%) by addition of Δ57–70 indicating the importance of the 47–49 region. A stimulatory effect of Δ57–70 was confirmed in the double deletion (Matα:Δ57–70;47–49) construct preceding the CO-GCSF. Secondary and tertiary structures, when predicted using I-TASSER, allowed to understand the relationship between structural changes and their impact on G-CSF secretion. The Δ57–70 amino acids form a major part of 3rd alpha-helix in the pre-pro peptide and its distortion increased the flexibility of the loop, thereby promoting its interaction with the cargo protein. A minimum loop length was found to be necessary for secretion. The strict control in the process of secretion appeared to be overcome by changing the secondary structures in the signal peptides. Such fine tuning can allow enhanced secretion of other therapeutics in this expression system. Conclusions Among the different truncations (Matα:Δ57–70, Matα:Δ47–49, Matα:Δ30–43, Matα:Δ57–70;30–43, Matα:Δ57–70;47–49) in pro-peptide of α-MAT secretion signal, Matα:Δ57–70 fused to CO-GCSF, led to highest G-CSF titre as compared to other Matα truncations. On the other hand, Matα:Δ30–43 and Matα:Δ47–49 fused to the WT-GCSF dampened the secretion of this protein indicating important role of these segments in the secretion of the cargo protein.

scholarly journals Proliferation Signaling and Activation of Shc, p21Ras, and Myc Via Tyrosine 764 of Human Granulocyte Colony-Stimulating Factor Receptor

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1924-1933 ◽  
Author(s):  
John P. de Koning ◽  
Amrita A. Soede-Bobok ◽  
Anita M. Schelen ◽  
Louise Smith ◽  
Daphne van Leeuwen ◽  
...  
Keyword(s):  
Distal Region ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Signaling Mechanisms ◽  
Fold Reduction ◽  
Multiple Signaling ◽  
Stimulating Factor

The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF–mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF–containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF–mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF–driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.

scholarly journals Proliferation Signaling and Activation of Shc, p21Ras, and Myc Via Tyrosine 764 of Human Granulocyte Colony-Stimulating Factor Receptor

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1924-1933 ◽  
Author(s):  
John P. de Koning ◽  
Amrita A. Soede-Bobok ◽  
Anita M. Schelen ◽  
Louise Smith ◽  
Daphne van Leeuwen ◽  
...  
Keyword(s):  
Distal Region ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Signaling Mechanisms ◽  
Fold Reduction ◽  
Multiple Signaling ◽  
Stimulating Factor

Abstract The membrane-distal region of the cytoplasmic domain of human granulocyte colony-stimulating factor receptor (G-CSF-R) contains four conserved tyrosine residues: Y704, Y729, Y744, and Y764. Three of these (Y729, Y744, and Y764) are located in the C-terminal part of G-CSF-R, previously shown to be essential for induction of neutrophilic differentiation. To determine the role of the tyrosines in G-CSF–mediated responses, we constructed tyrosine-to-phenylalanine (Y-to-F) substitution mutants and expressed these in a differentiation competent subclone of 32D cells that lacks endogenous G-CSF-R. We show that all tyrosines can be substituted essentially without affecting the differentiation signaling properties of G-CSF-R. However, substitution of one specific tyrosine, ie, Y764, markedly influenced proliferation signaling as well as the timing of differentiation. 32D cells expressing wild-type (WT) G-CSF-R (or mutants Y704F, Y729F, or Y744F) proliferated in G-CSF–containing cultures until day 8 and then developed into mature neutrophils. In contrast, 32D/Y764F cells arrested in the G1 phase of the cell cycle within 24 hours and showed complete neutrophilic differentiation after 3 days of culture. This resulted in an average 30-fold reduction of neutrophil production as compared with the 32D/WT controls. Importantly, G-CSF–mediated activation of Shc, p21Ras and the induction of c-myc were severely reduced by substitution of Y764. These findings indicate that Y764 of G-CSF-R is crucial for maintaining the proliferation/differentiation balance during G-CSF–driven neutrophil development and suggest a role for multiple signaling mechanisms in maintaining this balance.

scholarly journals The Expression and Role of Multiple Forms of Granulocyte Colony-Stimulating Factor in Medaka Fish

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 35-36
Author(s):  
Ayame Ogawa ◽  
Ryo Yamagishi ◽  
Takeru Matsumoto ◽  
Satoshi Ansai ◽  
Kiyoshi Naruse ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Severe Neutropenia ◽  
Granulocyte Colony Stimulating Factor ◽  
Medaka Fish ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Hematopoietic Growth Factors ◽  
Gene Expressions ◽  
Stimulating Factor

Diversity in the repertoires of granulocytes, such as neutrophils, basophils, and eosinophils, has been known among more than seventy thousand vertebrate species. Granulocyte colony-stimulating factor (G-CSF; csf3) affects the differentiation, survival, and proliferation of neutrophils. However, the function of G-CSF in many animal species whose repertoire of granulocytes differs from that of mammals is not clear. We, therefore, attempt to investigate G-CSF function in adult d-rR strain of Medaka fish (Oryzias latipes). Of the three types of granulocytes, Medaka fish possess only neutrophils. First of all, we assessed the distribution of neutrophils by detecting myeloperoxidase (MPO) -positive cells in the various organs, including the kidney, spleen, heart, liver, and intestine. Among those tissues, the cell proportion of neutrophils was the highest in the kidney (approx. 15%). We then identified the genome sequences of Medaka G-CSFa1x1 and G-CSFa1x2, as orthologs of the mammalian genes encoding G-CSF. They are transcriptional variants and located on chromosome 19. Phylogenetic analysis and locus analysis revealed that these sequences are classified as G-CSFa of teleost fish. The synteny and functional domains of the Medaka G-CSF receptor (G-CSFR; csf3r) is also conserved with those of other species of G-CSFR. They are different from typical mammalian G-CSFs because they have N-linked glycans. We compared the expression of Olcsf3a1 and Olcsf3r in each tissue and found higher expression in the spleen, kidney, and bone of the Medaka fish. The Olcsf3a1x2 expressed much higher than that of Olcsf3a1x1 in the kidney and spleen. In the Medaka exposed to breeding water containing lipopolysaccharide (LPS; Escherichia coli 055: B5), the number of peripheral neutrophils at 3 hours significantly elevated; while that of splenic neutrophils at 3 hours reduced, suggesting that splenic neutrophils were released to the circulation. The expression of Olcsf3a1 mRNA increased at 12 hours after LPS exposure in the bone The number of neutrophils in the kidney increased at 24 hours after LPS exposure, demonstrating that Olcsf3a1 stimulated myelopoiesis shortly after splenic neutrophil release. To elucidate the role of OlG-CSFa1 in a steady-state, we generated the Olcsf3a1 deficient Medaka (G-CSFa1-deficient) by CRISPR/Cas9 genome editing using short guide RNA for the shared sequence of both G-CSFa1x1 and G-CSFa1x2. As we expected, the heterozygous G-CSFa1-deficient Medaka exhibited severe neutropenia. The number of peripheral neutrophils significantly decreased to 1.80×109 cells/L, whereas that of wild-type Medaka was 14.2×109 cells/L. Remarkably, the number of peripheral erythrocytes displayed 2.91×1012 cells/L in the G-CSFa1-deficient Medaka, whereas that of the wild-type Medaka was 5.85×1012 cells/L. We, therefore, tested the gene expressions of hematopoietic growth factors and found that the relative gene expression of erythropoietin decreased in the kidney of G-CSFa1-deficient Medaka. The results show that G-CSF-deficient Medaka should be a valuable model to examine specific functions of G-CSF in producing granulocytes and its synergistic effect on erythropoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Characterization of recombinant-derived granulocyte-colony stimulating factor (G-CSF)

1988 ◽  
Vol 256 (1) ◽  
pp. 213-218 ◽  
Author(s):  
P Wingfield ◽  
R Benedict ◽  
G Turcatti ◽  
B Allet ◽  
J J Mermod ◽  
...  
Keyword(s):  
Coli Strain ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Disulphide Bonds ◽  
Disulphide Bond Formation ◽  
Stimulating Factor ◽  
Factor G

Human granulocyte colony-stimulating factor (G-CSF), and a mutant having a Ser for Cys substitution at residue 18 were produced in Escherichia coli strain W3110. About 60 mg of pure protein was obtained from 50 g of wet cells with a recovery of about 20%. The proteins were characterized physically and chemically, including determination of disulphide bonds, which were found to exist between residues 37-43 and 65-75. Cys-18 is not involved in disulphide bond formation and was substituted by Ser with no effects on gross protein conformation or biological activity. Both the wild-type and the mutant recombinant-derived proteins, although not glycosylated, possess colony-stimulating activities. In a bioassay using the murine myelomonocytic leukaemic cell line WEH1 3B D+, activities were obtained which were similar to those of natural G-CSF and of a glycosylated recombinant-derived human G-CSF produced in monkey cells.

P–391 Role of subcutaneous granulocyte colony-stimulating factor infusion in thin endometrium

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
K Banerjee ◽  
B Singla
Keyword(s):  
Pregnancy Rate ◽  
Clinical Pregnancy ◽  
P Value ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Thin Endometrium ◽  
Group 2 ◽  
Stimulating Factor ◽  
Group 1

Abstract Study question To assess the role of subcutaneous granulocyte colony-stimulating factor (G-CSF) in thin endometrium cases. Summary answer G CSF has beneficial role to improve the endometrium thickness in thin endometrium. What is known already Endometrium is very important for embryo implantation and the endometrial thickness is the marker of receptivity of the endometrium. Study design, size, duration Study design - Retrospective analysis Size - 88 infertile females with thin endometrium (< 7 mm) in the age group of 23 to 40 years Duration - one year. Participants/materials, setting, methods In the group 1 of 44 females, subcutaneous infusion of G CSF (300 mcg/ml) was added along with other supplements and if lining was not more than 7 mm in 72 hours, then second infusion was given. In the group 2 of 44 females, only estradiol valerate and sildenafil were given.The efficacy of G CSF was evaluated by assessing the endometrium thickness before embryo transfer, pregnancy rates and clinical pregnancy rates. Main results and the role of chance There was no difference between the two groups regarding demographic variables, egg reserve, sperm parameters, number of embryos transferred and embryo quality. . The pregnancy rate was 60% (24 out of 40 cases) in the group 1 that was significantly higher than in-group 2 that was 31% (9 out of 29 cases) with p value < 0.0001. The clinical pregnancy rate was also significantly higher in-group 1 (55%) as compared to group 2 (24%) with p value < 0.0001. Limitations, reasons for caution Further larger cohort studies are required to explore the subcutaneous role of G CSF in thin endometrium. Wider implications of the findings: Granulocyte colony-stimulating factor has beneficial role to improve the endometrium thickness in thin endometrium. In most of previous studies, the intrauterine infusion of G CSF was given to improve the uterine lining. This is one of the few studies done that showed subcutaneous role of G CSF in thin endometrium. Trial registration number Not applicable

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