Bacterial overexpression and purification of soluble recombinant human serum albumin using maltose-binding protein and protein disulphide isomerase

Nguyen, Minh Tan; Heo, Yunseok; Hang, Bich; Baek, Sang-Ki; Kim, Chong Jai; Jang, Yeon Jin; Lee, Weontae; Choe, Han

doi:10.1016/j.pep.2019.105530

Cited by 18 publications

(11 citation statements)

References 56 publications

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“…Minimal secretion of the recombinant albumin is achieved from Bacillus subtilis as higher levels of expression overwhelm the secretion pathway [40]. In Escherichia coli, rHSA tends to accumulate as unfolded, insoluble aggregates in inclusion bodies, requiring denaturation and refolding to obtain a suitably active product [19]. These factors are major bottlenecks that increase the number of purification steps, production cost, and highly stringent quality control to achieve the desired quality and quantity of recombinant HSA.…”

Section: Recombinant Hsamentioning

confidence: 99%

“…For all these applications, large quantities of HSA are classically sourced from blood serum. However, recombinant HSA from heterologous sources such as Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, Kluyveromyces lactis, transgenic animals, and plants have proven to be most beneficial for biotechnological purposes [14][15][16][17][18][19][20][21][22].…”

Section: Introductionmentioning

confidence: 99%

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Structural and Biochemical Features of Human Serum Albumin Essential for Eukaryotic Cell Culture

Mishra

Heath

2021

IJMS

115

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Serum albumin physically interacts with fatty acids, small molecules, metal ions, and several other proteins. Binding with a plethora of bioactive substances makes it a critical transport molecule. Albumin also scavenges the reactive oxygen species that are harmful to cell survival. These properties make albumin an excellent choice to promote cell growth and maintain a variety of eukaryotic cells under in vitro culture environment. Furthermore, purified recombinant human serum albumin is mostly free from impurities and modifications, providing a perfect choice as an additive in cell and tissue culture media while avoiding any regulatory constraints. This review discusses key features of human serum albumin implicated in cell growth and survival under in vitro conditions.

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Section: Recombinant Hsamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Structural and Biochemical Features of Human Serum Albumin Essential for Eukaryotic Cell Culture

Mishra

Heath

2021

IJMS

115

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“…In addition to these genes, SHuffle T7 expresses DsbC, which further alters the redox state of the cytoplasm [ 37 ]. In our previous studies, we found that the use of the SHuffle T7 or Origami2 (DE3) strains increased the solubility of the expressed passenger proteins [ 30 , 38 ]. Not surprisingly, this effect was more dramatic for albumin than for oncostatin M, which contain 17 and 2 disulfide bonds, respectively.…”

Section: Discussionmentioning

confidence: 99%

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain

Nguyen

et al. 2021

IJMS

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Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.

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“…hSCF164 has also been expressed in misfolded insoluble form in E. coli, resulting in inclu-sion bodies requiring complicated refolding processes to produce an active form [26,28,39]. MBP and PDIb'a' are two tags that have proven to be consistent effective solubilization enhancers [40,41]. Therefore, the two tags were tested for their effects on the solubility of the hSCF164.…”

Section: Discussionmentioning

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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Bacterial overexpression and purification of soluble recombinant human serum albumin using maltose-binding protein and protein disulphide isomerase

Cited by 18 publications

References 56 publications

Structural and Biochemical Features of Human Serum Albumin Essential for Eukaryotic Cell Culture

Structural and Biochemical Features of Human Serum Albumin Essential for Eukaryotic Cell Culture

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain

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

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