Expression, Refolding, And Purification Of Recombinant Human Stem Cell Factor And Determination Of Its Effect On Ex Vivo Expansion Of Cord Blood

Objectives:1) to obtain recombinant human stem cell factor (rhSCF) protein using an E.coli expression system, and to study the biological activity of rhSCF on ex vivo expansion of cord blood;2) to obtain a specific monoclonal antibody (MAb) to rhSCF and to determine the effects of the antibody on the biological activity of rhSCF.Methods:rhSCF was obtained from inclusion bodies following IPTG-induced expression in E.coli. Purified rhSCF was obtained after dialysis, refolding and CM-Sepharose chromatography. MNC was extracted from umbilical cord blood (UCB) via Ficoll density gradient centrifugation, and cells with CD34epitope were detected using the MACS kit. The biological activity of rhSCF was assessed using a previously established protocol; rhSCF was added together with thrombopoietin (TPO) and FLT-3ligand (FL) in a7-day ex vivo cord blood expansion assay. High-titer MAb to rhSCF was obtained following immunochemical screening of various MAb cell lines. The effect of the MAb on the biological activity of rhSCF was assessed through determination of the proportion of CD34+cells in the ex vivo expansion assay, using flow cytometry. The peptide mapping was used to identify the location of rhSCF’s active site.Results:Production of the rhSCF was readily induced by IPTG in the E.coli expression system. The purification of rhSCF from inclusion bodies after refolding and CM-Sepharose chromatography can be accomplished with high yield. The final product was electrophoretically homogeneous. The MAb cell line23C8was found to have a high titer and specificity for rhSCF. In ex vivo cord blood expansion assays, the addition of rhSCF to assays containing FL and TPO led to further stimulation of the expansion of CD34+cells. The ability of rhSCF to stimulate the expansion of CD34’cells was significantly inhibited by the23C8anti-rhSCF MAb. The active site of rhSCF was located in the93th-165th amino acid sequence.Conclusion:A reproducible and efficient prokaryotic expression system for rhSCF, along with highly effective purification and refolding procedures, was established. The rhSCF protein produced had biological activities comparable to that of a reference rhSCF preparation. The MAb cell line23C8produces high-titer, specific anti-rhSCF MAb, which appears to bind directly to parts of rhSCF that are critical for biological activity, as the MAb effectively prevented rhSCF-stimulated ex vivo expansion of CD34’cells. The active site of rhSCF was located in the93th-165th amino acid sequence. Overall, this study laid a solid foundation for further research on the optimization of ex vivo expansion of cord blood.