title: Cloning, expression and identification of an isoform of human stromal cell derived factor-1α.
abstract: Human stromal cell derived factor-1α (hSDF-1α), a chemotactic factor of stem cells, regulates inflammation, promotes the mobilization of stem cells and induces angiogenesis following ischemia. Six SDF-1 isoforms, SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1ϕ, which all contain a signal peptide at the N-terminus, have been reported. In the present study a special isoform of hSDF-1α is described that does not contain the N-terminal signal peptide sequence. ThehSDF-1αgene was cloned with the recombinant plasmid pCMV-SPORT6-hSDF1 as the template, and the prokaryotic expression vector pET15b-hSDF-1α was constructed. This hSDF-1α was successfully expressed as an inclusion body inEscherichia coliBL21(DE3). The recombinant hSDF-1α was refoldedin vitroand separated by cation exchange chromatography. Following these two steps the purity of the hSDF-1α was able to reach >85%. The recombinant hSDF-1α was then purified by size-exclusion chromatography. SDS-PAGE analysis demonstrated that the purity of the hSDF-1α was >95%, which meets almost all the requirements of a protein experiment. Chemotactic activity of the recombinant hSDF-1α was analyzed by Transwell migration assay and it was found that the recombinant hSDF-1α was able to stimulate THP-1 cell migration. These data suggest that the procedure of producing recombinant hSDF-1α proteins with chemotactic activity was feasible and the N-terminal signal peptide of hSDF-1α has little effect on the chemotactic activity of hSDF-1α.
authors.affiliation: College of Life Science, Northwest University, Xi'an, Shaanxi 710069, P.R. China ; College of Biological Science and Engineering, Shaanxi University of Technology, Hanzhong, Shaanxi 723000, P.R. China.
authors.forename: Yin-Ku
authors.initials: YK
authors.lastname: Liang
authors.affiliation: College of Life Science, Northwest University, Xi'an, Shaanxi 710069, P.R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ping
authors.affiliation: College of Life Science, Northwest University, Xi'an, Shaanxi 710069, P.R. China.
authors.forename: Liu-Jiao
authors.initials: LJ
authors.lastname: Bian
keywords: chemotactic activity; cloning; human stromal cell derived factor-1α; purification