Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner

Bals, C.; Schambach, Axel; Meyer, Johann; Scheper, Thomas; Rinas, Ursula

doi:10.1016/j.jbiotec.2011.01.012

Cited by 8 publications

(5 citation statements)

References 45 publications

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“…It is unknown how His-hSCF248 is so soluble. Usually, the effect of EC 50 of hSCF164 on TF-1 cell proliferation is reported to be~10 ng/mL [31][32][33]. Consistent with these results, our purified hSCF164 and commercial hSCF189 showed EC 50 values of 2-10 ng/mL.…”

Section: Discussionsupporting

confidence: 88%

“…Most companies selling hSCF show the biological activities of their products using TF-1 cell proliferation. Furthermore, many researchers have used TF-1 cells to test the activity of hSCF [31][32][33]. Therefore, we chose TF-1 cells for our assay system.…”

Section: Discussionmentioning

confidence: 99%

“…These denatured proteins require a cumbersome solubilization and refolding process, and, even after refolding, the biological activity can be attenuated. Thioredoxin has been used as a fusion partner or co-expressed for the soluble expression of hSCF164 in E. coli cytoplasm [31,32]. structure of the His-hSCF248, His-hSCF164, PDIb'a'-hSCF164, and MBP-hSCF164 constructs.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Lee

Paula

Baek

et al. 2021

IJMS

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“…Specifically, solubility was addressed by the cloning of the Tgu6.1 gene into thioredoxin containing pET-32a XA/LIC vector using ligation independent cloning (LIC) ( Figure 2 C). Thioredoxin as a fusion partner has been shown to significantly increase the solubility of proteins synthesized in the E. coli cytoplasm [ 30 , 45 , 46 , 47 , 48 ]. Furthermore, when expressed in an oxidizing environment, thioredoxin has been observed to catalyze disulfide bond formation [ 49 ].…”

Section: Resultsmentioning

confidence: 99%

Characterization and Recombinant Expression of Terebrid Venom Peptide from Terebra guttata

Moon

Gorson

Wright

et al. 2016

Toxins

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Venom peptides found in terebrid snails expand the toolbox of active compounds that can be applied to investigate cellular physiology and can be further developed as future therapeutics. However, unlike other predatory organisms, such as snakes, terebrids produce very small quantities of venom, making it difficult to obtain sufficient amounts for biochemical characterization. Here, we describe the first recombinant expression and characterization of terebrid peptide, teretoxin Tgu6.1, from Terebra guttata. Tgu6.1 is a novel forty-four amino acid teretoxin peptide with a VI/VII cysteine framework (C–C–CC–C–C) similar to O, M and I conotoxin superfamilies. A ligation-independent cloning strategy with an ompT protease deficient strain of E. coli was employed to recombinantly produce Tgu6.1. Thioredoxin was introduced in the plasmid to combat disulfide folding and solubility issues. Specifically Histidine-6 tag and Ni-NTA affinity chromatography were applied as a purification method, and enterokinase was used as a specific cleavage protease to effectively produce high yields of folded Tgu6.1 without extra residues to the primary sequence. The recombinantly-expressed Tgu6.1 peptide was bioactive, displaying a paralytic effect when injected into a Nereis virens polychaete bioassay. The recombinant strategy described to express Tgu6.1 can be applied to produce high yields of other disulfide-rich peptides.

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“…The purification of biotechnological products from complex sources like fermentation broth [10][11][12][13], cell culture supernatants [14], or natural sources [15][16][17] is the major task of downstream processing. Since affinity-based separations are regarded to be the finest available [18], they are especially useful for the purification of biopharmaceuticals.…”

Section: Introductionmentioning

confidence: 99%

Aptamers as affinity ligands for downstream processing

Walter

Stahl

Scheper

2012

Engineering in Life Sciences

Self Cite

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Aptamers are single‐stranded synthetic oligonucleotides that are able to capture their target molecule with high affinity and specificity. Therefore, they can be thought of as nucleic acid‐based alternative to antibodies, which have several advantages over their amino acid‐based counterparts. Consequently, aptamers can be used in different applications based on molecular recognition including affinity separations. This review will summarize the state‐of‐the‐art in aptamer‐based affinity separations; will discuss the current limitations and will highlight possible future prospects. The first part will point out the advantages of aptamers in downstream processing. Here, the properties of aptamers will be discussed along with their implications on downstream processing from a user's point of view. In the second part, a brief summary of the literature is given with focus on aptamer‐based separation of proteins. Finally, some drawbacks of aptamers will be illustrated and possibilities to overcome these limitations will be suggested. New technologies in the fields of aptamer selection and synthesis are expected to further promote the use of aptamers as affinity ligands in downstream processing.

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Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner

Cited by 8 publications

References 45 publications

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

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

Characterization and Recombinant Expression of Terebrid Venom Peptide from Terebra guttata

Aptamers as affinity ligands for downstream processing

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