Cloning, expression and identification of an isoform of human stromal cell derived factor-1α

Abstract

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. The hSDF-1α gene 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 in Escherichia coli BL21(DE3). The recombinant hSDF-1α was refolded in vitro and 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.

Evaluation of stromal cell-derived factor-1β Gene polymorphism G801A among Egyptian patients with type 2 diabetes mellitus

“…6 Six SDF-1 isoforms have been reported SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1ϕ where all share a common Nterminal amino acid sequence, but a distinct Cterminus. 7 SDF-1 possesses diverse physiological and biochemical functions. 8 It is considered to be a homeostatic chemokine, 6 and SDF-1 plays a particularly important role in directing T cell migration and therefore immune processes.…”