One-step metal affinity purification of recombinant hFGF19 without using tags

Choi, Hyun-Woo; Cheong, Dae-Eun; Yoo, SooNam; Kim, Geun-Joong

doi:10.1016/j.pep.2022.106186

Cited by 6 publications

(8 citation statements)

References 26 publications

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“…NIH3T3 cells were treated with the purified cp‐hFGF7 115‐114 , and ERK1/2 phosphorylation in these cells was evaluated in a time‐ and dose‐dependent manner. [ 44 ] As shown in Figure 4A, cp‐hFGF7 115‐114 (50 ng mL −1 ) induced clear phosphorylation of ERK1/2, and its effect decreased in a time‐dependent manner. In addition, cp‐hFGF7 115‐114 also activated ERK1/2 phosphorylation in a dose‐dependent manner that was similar to the commercially available wild‐type protein (Figure 4B).…”

Section: Resultsmentioning

confidence: 94%

Construction and characterization of a functional variant hFGF7 with enhanced properties by circular permutation

Choi,

Lee,

Cheong

et al. 2024

Biotechnology Journal

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Human fibroblast growth factor 7 (hFGF7) is a member of the paracrine‐acting FGF family and mediates various reactions such as wound healing, tissue homeostasis, and liver regeneration. These activities make it a plausible candidate for pharmaceutical applications as a drug. However, the low expression level and stability of the recombinant hFGF7 were known to be major hurdles for further applications. Here, the expression level and stability of hFGF7 were attempted to improve by changing the order of amino acids through circular permutation (CP), thereby expecting an alternative fate according to the N‐end rule. CP‐hFGF7 variants were constructed systematically by using putative amino acid residues in the loop region that avoided the disruption of the structural integrity especially in the functional motif. Among them, cp‐hFGF7115‐114 revealed a relatively higher expression level in the soluble fraction than the wild‐type hFGF7 and was efficiently purified (7 mg L−1) to apparent homogeneity. The activity and stability of the purified variant cp‐hFGF7115‐114 were comparable or superior to that of the wild‐type hFGF7, thereby strongly suggesting that CP could be an alternative tool for the functional expression of hFGF7 in Escherichia coli.

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Section: Resultsmentioning

confidence: 94%

Construction and characterization of a functional variant hFGF7 with enhanced properties by circular permutation

Choi,

Lee,

Cheong

et al. 2024

Biotechnology Journal

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“…We produced tag-free recombinant FGF19 and Aldafermin (Choi et al , 2023, 2020) (Figure 3A) and performed intra-peritoneal injections of 2μg of purified proteins per mouse (corresponding to the 3 mg of the drug given to patients in clinical trials(Harrison et al , 2018)). Transcriptional repression of hepatic Cyp7a1 , a bona fide target of FGF19, confirmed the biological activity of the recombinant proteins in vivo (Figure 3B).…”

Section: Resultsmentioning

confidence: 99%

“…The plasmids for the bacterial expression of FGF19 and Aldafermin were generated by producing a sequence composed of the T5 promoter followed by the sequence coding for the disulfide bond isomerase (ΔssDsbC), then a T7 promoter followed by a codon-optimized variant of either FGF19 or Aldafermin CDS based on published work by Choi et al (Choi et al , 2023). Those sequences were subsequently cloned in the pQLinkG2 (Addgene #13671) using the XhoI/PasI restriction sites.…”

Section: Methodsmentioning

confidence: 99%

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FGF19 and its analog Aldafermin cooperate with MYC to induce aggressive hepatocarcinogenesis

Ursic-Bedoya,

Desandré,

Chavey

et al. 2023

Preprint

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FGF19 hormone has pleiotropic metabolic functions, notably the modulation of insulin sensitivity, glucose/lipid metabolism and energy homeostasis. On top of its physiological metabolic role, FGF19 has been identified as a potentially targetable oncogenic driver, notably in hepatocellular carcinoma (HCC). Nevertheless, FGF19 remained an attractive candidate for treatment of metabolic disease, prompting the development of analogs uncoupling its metabolic and tumor-promoting activities. Using pre-clinical mice models of somatic mutation driven HCC, we assessed the oncogenicity of FGF19 in combination with frequent HCC tumorigenic alterations: p53 inactivation, CTNNB1 mutation, CCND1 or MYC overexpression. Our data revealed a strong oncogenic cooperation between FGF19 and MYC. Most importantly, we show that this oncogenic synergy is conserved with a FGF19-analog Aldafermin (NGM282), designed to solely mimic the hormone's metabolic functions. In particular, even a short systemic treatment with recombinant proteins triggered rapid appearance of proliferative foci of MYC-expressing hepatocytes. The fact that FGF19 analog Aldafermin is not fully devoid of the hormone's oncogenic properties raises concerns in the context of its potential use for patients with damaged, mutation-prone liver.

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“…The plasmids for the bacterial expression of FGF19 and Aldafermin were generated by producing a sequence composed of the T5 promoter followed by the sequence coding for the disulfide bond isomerase (∆ssDsbC), then a T7 promoter followed by a codon-optimized variant of either FGF19 or Aldafermin CDS based on published work by Choi et al ( 2023 ). Those sequences were subsequently cloned in the pQLinkG2 (Addgene #13671) using the XhoI/PasI restriction sites.…”

Section: Methodsmentioning

confidence: 99%

FGF19 and its analog Aldafermin cooperate with MYC to induce aggressive hepatocarcinogenesis

Ursic-Bedoya,

Desandré,

Chavey

et al. 2024

EMBO Mol Med

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FGF19 hormone has pleiotropic metabolic functions, including the modulation of insulin sensitivity, glucose/lipid metabolism and energy homeostasis. On top of its physiological metabolic role, FGF19 has been identified as a potentially targetable oncogenic driver, notably in hepatocellular carcinoma (HCC). Nevertheless, FGF19 remained an attractive candidate for treatment of metabolic disease, prompting the development of analogs uncoupling its metabolic and tumor-promoting activities. Using pre-clinical mice models of somatic mutation driven HCC, we assessed the oncogenicity of FGF19 in combination with frequent HCC tumorigenic alterations: p53 inactivation, CTNNB1 mutation, CCND1 or MYC overexpression. Our data revealed a strong oncogenic cooperation between FGF19 and MYC. Most importantly, we show that this oncogenic synergy is conserved with a FGF19-analog Aldafermin (NGM282), designed to solely mimic the hormone’s metabolic functions. In particular, even a short systemic treatment with recombinant proteins triggered rapid appearance of proliferative foci of MYC-expressing hepatocytes. The fact that FGF19 analog Aldafermin is not fully devoid of the hormone’s oncogenic properties raises concerns in the context of its potential use for patients with damaged, mutation-prone liver.

show abstract

One-step metal affinity purification of recombinant hFGF19 without using tags

Cited by 6 publications

References 26 publications

Construction and characterization of a functional variant hFGF7 with enhanced properties by circular permutation

Construction and characterization of a functional variant hFGF7 with enhanced properties by circular permutation

FGF19 and its analog Aldafermin cooperate with MYC to induce aggressive hepatocarcinogenesis

FGF19 and its analog Aldafermin cooperate with MYC to induce aggressive hepatocarcinogenesis

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