[Expression, purification, and characterization of the first three immunoglobulin-like domains of human stem cell factor receptor].

OBJECTIVE: To express the first three immunoglobulin-like domains of human stem cell factor receptor (c-Kit/Ig1-3) in E. coli and HEK293 ET cells and study their binding activity for stem cell factor (SCF). METHODS: In prokaryotic expression system, a double mutant form of c-Kit /Ig1-3 (c-Kit /Ig1-3(DM) was produced by overlap PCR and cloned into pET16b. The recombinant protein was expressed in E. coli BL21 (DE3) and refolded by dilution. In eukaryotic expression system, the gene of c-Kit/Igl13 with eight histidine segments was cloned into pEAK12 and the recombinant plasmid was transfected into HEK293 ET cells. The fusion protein was harvested from the growth medium and purified on Ni-NTA agarose column. The recombinant protein was tested for the receptor binding activity with his-tag pull-down and enzyme-linked immunosorbent binding assay. RESULTS: In E. coli c-Kit /Ig1-3(DM) as produced as an inclusion body and showed low binding activity for SCF after refolding. Two HEK293 ET cell clones that express high levels of c-Kit/Ig1-3 were produced and each clone secreted 2p micro/ml of recombinant protein, whose relative molecular mass was about 58,000. Eukaryotically expressed c-Kit/Ig1-3 had specific binding activity for SCF, and the dissociation constant (Kd) was 9.39 nmol/L. CONCLUSION: c-Kit/Ig1-3 with high receptor binding activity is successfully produced in HEK293 ET cells.

Organophosphorus flame retardants and plasticizers: sources, occurrence, toxicity and human exposure.

Due to the restricted use and ban of brominated flame retardants, organophosphorus compounds (OPs), extensively used as flame retardants and plasticizers, are ubiquitous in various environmental compartments worldwide. The present study shows that the release of OPs from a wide variety of commercial products and wastewater discharge might be considered as primary emission sources and that high potential of long-range atmospheric transport and persistence of OPs would be responsible for their presence in various matrices on a global scale. The occurrence and environmental behaviors of OPs in diverse matrices (e.g., dust, air, water, sediment, soil and biota) are reviewed. Human exposures to OPs via dermal contact, dust ingestion, inhalation and dietary intake are comprehensively evaluated. Finally, this study identifies gaps in the existing issues and generates a future agenda for the emerging contaminants OPs.

High-level expression of human stem cell factor fused with erythropoietin mimetic peptide in Escherichia coli.

Stem cell factor (SCF) and erythropoietin are essential for normal erythropoiesis and induce proliferation and differentiation synergistically for erythroid progenitor cells. Here, we report our work on construction of SCF/erythropoietin mimetic peptide (EMP) fusion protein gene, in which human SCF cDNA (1-165aa) and EMP sequence (20aa) were connected using a short (GGGGS) or long (GGGGSGGGGGS) linker sequence. The SCF/EMP gene was cloned into the pBV220 vector and expressed in the Escherichia coli DH5alpha strain. The expression level of the fusion protein was about 30% of total cell protein. The resulting inclusion bodies were solubilized with 8 M urea, followed by dilution refolding. The renatured protein was subsequently purified by Q-Sepharose FF column. The final product was >95% pure by SDS-PAGE and the yield of fusion protein was about 40 mg/L of culture. UT-7 cell proliferation and human cord blood cell colony-forming assays showed that the fusion proteins exhibited more potent activity than recombinant human SCF, suggesting a new strategy to enhance biological activities of growth factors.