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

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Human stromal cell derived factor-1α (hSDF-1α) is a key factor in stem cell mobilization, inflammation regulation, and angiogenesis.
• Six known isoforms of SDF-1 exist, all typically possessing an N-terminal signal peptide.
• A unique hSDF-1α isoform lacking this signal peptide was investigated.

Purpose of the Study:

• To clone, express, and purify a recombinant hSDF-1α isoform without the N-terminal signal peptide.
• To assess the chemotactic activity of this novel hSDF-1α isoform.
• To determine the effect of the N-terminal signal peptide on hSDF-1α's chemotactic function.

Main Methods:

• Cloning of the hSDF-1α gene using pCMV-SPORT6-hSDF1 and construction of the pET15b-hSDF-1α prokaryotic expression vector.
• Expression of recombinant hSDF-1α in Escherichia coli BL21(DE3) as inclusion bodies, followed by in vitro refolding.
• Purification using cation exchange chromatography and size-exclusion chromatography, with purity assessed by SDS-PAGE.
• Chemotactic activity evaluation via Transwell migration assay using THP-1 cells.

Main Results:

• Recombinant hSDF-1α was successfully expressed and purified to >95% purity.
• The purified recombinant hSDF-1α exhibited significant chemotactic activity, stimulating THP-1 cell migration.
• The absence of the N-terminal signal peptide did not substantially impair the protein's chemotactic function.

Conclusions:

• A feasible method for producing recombinant hSDF-1α proteins with chemotactic activity was established.
• The N-terminal signal peptide appears to have a minimal role in the chemotactic activity of hSDF-1α.
• This research provides insights into SDF-1α isoform function and production for experimental use.