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

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 6x His tag, was expressed in Escherichia coli as inclusion bodies after isopropyl beta-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 microg/ml of rhDSL. The rhDSL protein might therefore be a potential supplement for the expansion of UCB-primitive hematopoietic cells.

Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in Escherichia coli using different carbon sources and chromatography sequences

“…Very few articles report the use of ion exchange before metal affinity and, in general, they did not discuss the reasons for adopting this sequence. Zhao et al (2010) states that cation exchange is efficient for LPS removal and Schmidt et al (2007) attributes the low recovery of Histagged protein to a change in oxidation state or to a complex formation between the Ni 2+ resin and yeast intracellular contents when large volumes of crude yeast cell lysate is applied to Ni 2+ resin. Here, the sequence anion exchange followed by metal affinity was chosen since the binding capacity of IMAC-Sepharose was greatly diminished after five performances as first chromatography step (not shown);…”

Recombinant expression and characterization of biologically active protein delta homolog 1

“…The N-terminal part of human Delta-like 1 (DLL1) was expressed as GST-tagged fusion protein and refolded with chromatographic methods [36]. In another approach, the same protein was expressed as His-tagged fusion protein and refolded by rapid dilution [37]. In these two publications, the recombinant DLL1 proteins consist of just one DSL domain, which itself contains 6 cysteines to form 3 disulfide bonds.…”

High-level expression and characterization of bioactive human truncated variant of hepatocyte growth factor in Escherichia coli

“…A number of studies have demonstrated that the 69 His tag had no effect on the activity of target protein, including enzyme (Zhou et al 2010), cytokine (Cao et al 2005), antibody (Cao et al 2006), transcription factor (Zhao et al 2010), and vaccine (Villaflores et al 2013).…”