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

Liang,Yin‑Ku; Ping,Wei; Bian,Liu‑Jiao

doi:10.3892/etm.2015.2355

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

Authors:
- Yin‑Ku Liang
- Wei Ping
- Liu‑Jiao Bian
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Affiliations: College of Life Science, Northwest University, Xi'an, Shaanxi 710069, P.R. China
Published online on: March 16, 2015 https://doi.org/10.3892/etm.2015.2355
Pages: 1751-1756

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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.

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