A novel recombinant dual human SCF expressed in and purified from silkworm, <i>Bombyx mori</i>, possesses higher bioactivity than recombinant monomeric human SCF

Han, Junhai; Zang, Yuhui; Lu, Haiqin; Zhu, Jie; Qin, Jianqi

doi:10.1111/j.1600-0609.2004.00221.x

Cited by 6 publications

(2 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The linker peptide mimicked the hinge region of mouse IgG2b (GGGGSPGEPSGPISTINPSPPSKESHKSPNM) [30] and was relatively non‐antigenic determinant and was flexible enough to enable TPO and moieties to ‘wiggle’ freely and maintain their three‐dimensional structures. In our previous studies [31,32], we had successfully constructed SCF–M‐CSF (macrophage colony‐stimulating factor) and dual SCF fusion proteins respectively with a structurally similar linker peptide (GGGGSGGGGSGG), and showed, using binding‐affinity assays, that both moieties of the fusion proteins could bind to their receptors independently. In the case of TPO–SCF, we added a proline residue in the linker peptide.…”

Section: Discussionmentioning

confidence: 99%

A novel thrombopoietin–stem‐cell factor fusion protein possesses enhanced potential in stimulating megakaryocyte proliferation and differentiation

Zang¹,

Zhang²,

Wei³

et al. 2007

Biotech and App Biochem

Self Cite

View full text Add to dashboard Cite

TPO (thrombopoietin) and SCF (stem-cell factor) are functionally related cytokines with overlapping but distinct haematopoietic effects. In the present study, a novel TPO-SCF fusion protein that combined the complementary biological effects of TPO and SCF into a single molecule was expressed in, and purified from, Sf9 [Spodoptera frugiperda (fall armyworm)] insect cells. The specific activity of rhTPO (recombinant human TPO)-SCF in megakaryoblastic Mo7e cell proliferation assays was 2.90+/-0.35 x 10(7) units/micromol, approx. 1.7 times as high as that of rhTPO. The specific activity of rhTPO-SCF in TF-1 cells proliferation assays was 7.10+/-0.95 x 10(6) units/micromol, approx. 1.2 times as high as that of rhSCF (recombinant human SCF). In a megakaryocyte-colony-forming assay using human peripheral-blood CD34(+) cells, the SCF moiety of rhTPO-SCF worked in a synergistic way to augment the colony number and exhibited a higher potential to stimulate megakaryocyte colony growth. According to the results of EMSA (electrophoretic mobility-shift assay) and semi-quantitative RT (reverse transcriptase)-PCR, the synergistic effects of the SCF moiety were also reflected in increased STAT5 (signal transducer and activator of transcription 5) DNA binding and enhanced up-regulation of p21 expression in Mo7e cells treated by rhTPO-SCF, suggesting that rhTPO-SCF could be more potent in promoting megakaryocyte proliferation and differentiation.

show abstract

Section: Discussionmentioning

confidence: 99%

A novel thrombopoietin–stem‐cell factor fusion protein possesses enhanced potential in stimulating megakaryocyte proliferation and differentiation

Zang¹,

Zhang²,

Wei³

et al. 2007

Biotech and App Biochem

Self Cite

View full text Add to dashboard Cite

show abstract

“…Many studies of hSCF production have focused on hSCF164. In early studies, eukaryotic expression systems were used in an attempt to produce hSCF164 [22][23][24]. However, difficulties remain in the use of this system and in the comparatively high cost.…”

Section: Introductionmentioning

confidence: 99%

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

Lee

Paula

Baek

et al. 2021

IJMS

View full text Add to dashboard Cite

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.

show abstract

Silkworm expression system as a platform technology in life science

Kato

Kajikawa

Maenaka

et al. 2009

Appl Microbiol Biotechnol

178

110

View full text Add to dashboard Cite

Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3–6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the “Silkroad,” we expect that the “Bioroad” from Asia to Europe will be established by the silkworm expression system.

show abstract

A novel recombinant dual human SCF expressed in and purified from silkworm, Bombyx mori, possesses higher bioactivity than recombinant monomeric human SCF

Cited by 6 publications

References 31 publications

A novel thrombopoietin–stem‐cell factor fusion protein possesses enhanced potential in stimulating megakaryocyte proliferation and differentiation

A novel thrombopoietin–stem‐cell factor fusion protein possesses enhanced potential in stimulating megakaryocyte proliferation and differentiation

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

Silkworm expression system as a platform technology in life science

Contact Info

Product

Resources

About