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Expression, Purification, and Characterization of a Novel Soluble Form of Human Delta-like-1

Author:
Zhao, Mei, Wu, Mingyuan, Guo, Lingchen, Jiang, Junfen, Huang, Weiwei, Lin, Xiaojuan, Zhang, Zhonghui, Xiang, Di, Lu, Huili, Zhu, Shunying, Yu, Yan, Moldenhauer, Anja, Han, Wei
Source:
Applied biochemistry and biotechnology 2010 v.160 no.5 pp. 1415-1427
ISSN:
0273-2289
Subject:
Escherichia coli, affinity chromatography, anion exchange, cell differentiation, humans, inclusion bodies, signal transduction, stem cells, umbilical cord
Abstract:
The notch signaling pathway plays an important role in inhibiting cell differentiation and enhancing the repopulation capability of hematopoietic stem/progenitor cells. In this study, we developed rhDSL, a novel soluble form of Notch ligand Delta-like-1, which contains the DSL domain and the N-terminal sequence of the ligand, and investigated its function in ex vivo expansion of human umbilical cord blood (UCB)-primitive hematopoietic cells. The coding sequence for rhDSL was cloned into a pQE30 vector, and the recombinant rhDSL, fused with a 6× His tag, was expressed in Escherichia coli as inclusion bodies after isopropyl β-d-thiogalactoside induction. After renaturing by dilutions, the protein was purified through anion exchange followed by affinity chromatography. The purity of rhDSL protein was more than 99% with very low endotoxin. In combination with human c-kit ligand, the effect of rhDSL on ex vivo expansion of UCB CD34⁺ cells was found to be optimal at 1.5 μg/ml of rhDSL. The rhDSL protein might therefore be a potential supplement for the expansion of UCB-primitive hematopoietic cells.
Agid:
638783