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

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Abstract
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.
Author(s)
김종재노바이스 데 파올라 미셀따 후인 낌 칸백상기웬 민 탄이은영장연진정택현최한석
Issued Date
2021
Type
Article
Keyword
hSCF164hSCF248human stem-cell factorMBPrecombinant proteinsoluble protein
DOI
10.3390/ijms22126361
URI
https://oak.ulsan.ac.kr/handle/2021.oak/8017
https://ulsan-primo.hosted.exlibrisgroup.com/primo-explore/fulldisplay?docid=TN_cdi_doaj_primary_oai_doaj_org_article_8b1d3461f29b4a6cabd5d81d9b8c4b77&context=PC&vid=ULSAN&lang=ko_KR&search_scope=default_scope&adaptor=primo_central_multiple_fe&tab=default_tab&query=any,contains,Novel%20Bacterial%20Production%20of%20Two%20Different%20Bioactive%20Forms%20of%20Human%20Stem-Cell%20Factor&offset=0&pcAvailability=true
Publisher
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Location
스위스
Language
영어
ISSN
1422-0067
Citation Volume
22
Citation Number
12
Citation Start Page
0
Citation End Page
0
Appears in Collections:
Medicine > Medicine
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