Large‐scale purification and characterization of recombinant human stem cell factor in <i>Escherichia coli</i>

Chen, Lianghua; Cai, Feng; Zhang, Dan-ju; Zhang, Li; Zhu, Peng; Gao, Sheng

doi:10.1002/bab.1517

Cited by 6 publications

(5 citation statements)

References 35 publications

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“…However, specific E. coli strains expressing T7 polymerase are required and inducer concentrations (IPTG, Arabinose) may need to be tuned to mitigate potential negative effects associated with the high viral polymerase activity . Temperature-inducible protein expression vectors (e.g., pBV220, pSW2, pKF396M-5) are convenient because only shifting the culture temperature is required. − However, most established vectors are only available for sticky-end cloning with the limitations described above. Also, a fixed induction temperature is recommended for protein expression which may be problematic in some cases. − …”

Section: Introductionmentioning

confidence: 99%

Three Novel Escherichia coli Vectors for Convenient and Efficient Molecular Biological Manipulations

Gao

Hart

et al. 2018

J. Agric. Food Chem.

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We have constructed novel plasmids pANY2, pANY3, and pANY6 for flexible cloning with low false positives, efficient expression, and convenient purification of proteins. The pANY2 plasmid can be used for efficient isopropyl-β-d-thiogalactoside (IPTG) induced protein expression, while the pANY3 plasmid can be used for temperature-induced expression. The pANY6 plasmid contains a self-cleaving elastin-like protein (ELP) tag for purification of recombinant protein by simple ELP-mediated precipitation steps and removal of the ELP tag by self-cleavage. A urea-based denaturation and refolding processes for renaturation of insoluble inclusion bodies can be conveniently integrated into the ELP-mediated precipitation protocol, removing time-consuming dialysis steps. These novel vectors, together with the described strategies of gene cloning, protein expression, and purification, may have wide applications in biosciences, agricultural, food technologies, and so forth.

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Section: Introductionmentioning

confidence: 99%

Three Novel Escherichia coli Vectors for Convenient and Efficient Molecular Biological Manipulations

Gao

Hart

et al. 2018

J. Agric. Food Chem.

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“…Analisis literatur menunjukkan bahwa banyak hal yang harus diperhatikan dalam produksi rekombinan protein sebagai vaksin, atau sebagai tujuan terapeutik dan diagnosis. Salah satu sukses yang paling utama dalam aplikasi molekul biosimilar tersebut adalah bila berhasil mengembangkan produk tersebut dalam skala besar (Brown et al, 2014 ;Bustos et al, 2016 ;Chen et al, 2016).…”

Section: Pendahuluanunclassified

Pengaruh Jumlah Inokulum Sel Inang Bakteri E.coli BL21 (DE3) pLysS dan Waktu Overekspresi pada Produksi Protein Rekombinan Fim-C Salmonella typhi

Nurjayadi

Chairinnisa

Mentari

et al. 2018

JKV

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Recombinant protein Fim-C S.typhi is a potential protein that can be used as an alternative typhoid vaccine and produced on a large scale. However, before entering into a large-scale stage, laboratory optimum data on the factors that affect the production process of Fim-C Salmonella typhi proteins are required. This study aims to obtain information regarding the effect of host cell number E.coli BL21 (DE3) pLysS and overexpression time on production of recombinant protein Fim-C Salmonella typhi as the basis for developing candidate of typhoid vaccine. The optimization process of overexpression was done by adding 0.5 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) inducer into bacterial cultures of 2%, 4%, and 6% with 4, 5, and 6 hours over expression. The measurement of the concentration of Fim-C recombinant protein extracted samples were done by a spectrophotometric method used BCA Kit Assay Thermo ScientificTM with wavelength 590nm. The characterization of those protein extracts was performed using SDS-PAGE method. The results from the study concluded that the number of 4% E.coli bacterial cells and four-hour overexpression time are the optimum condition of Fim- C Salmonella typhi characterized by the presence of high-intensity bands at ± 31 kDa.

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“…However, difficulties remain in the use of this system and in the comparatively high cost. For the more economic and efficient production of hSCF164, heterologous expression using Escherichia coli has been adopted [13,[25][26][27][28][29][30]. However, hSCF164 expressed in E. coli resulted in denatured proteins and inclusion bodies.…”

Section: Introductionmentioning

confidence: 99%

Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor

Lee

Paula

Baek

et al. 2021

IJMS

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Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b’a’ domain (PDIb’a’) tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.

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Large‐scale purification and characterization of recombinant human stem cell factor in Escherichia coli

Cited by 6 publications

References 35 publications

Three Novel Escherichia coli Vectors for Convenient and Efficient Molecular Biological Manipulations

Three Novel Escherichia coli Vectors for Convenient and Efficient Molecular Biological Manipulations

Pengaruh Jumlah Inokulum Sel Inang Bakteri E.coli BL21 (DE3) pLysS dan Waktu Overekspresi pada Produksi Protein Rekombinan Fim-C Salmonella typhi

Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor

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