Identification of a Potential Modification Site in Human Stromal Cell-Derived Factor-1

Selective modification of proteins is an important tool to study their function However, it is still challenging to identify the best position to avoid a loss of activity By using a 6-nitroveratryl (Nvoc)-modification approach, we facilitate the identification of a potential modification sit Nvoc can be removed in situ by UV irradiation and accordingly allows directly the comparison of the biological activity of the modified and the unmodified protein derived from the same precursor As a test system, we vied stromal cell-derived factor-1 (SDF-1), which is involved in a wide range of physiological functions, mainly hematopoiesis and embryonic organ development This chemokine is a potential candidate in regenerative medicine because of its capability to attract stem cells to distinct localizations First, we synthesized the wildtype and he Nvoc-modified C-terminal segments SDF-1(50 68) and studied their secondary structure formation by circular dichroism spectroscopy By using the intern-mediated purification with a affinity chitin binding tag system, we then expressed the peptide thioester M-[A(49)]-SDF 1(1-49)-MESNA recombinantly, in which the valine at position 49 was replaced by a more suitable alanine residue to allow improved cleavage and ligation After ligation and refolding, the biological activity was proven in a cell-based inositol phosphate accumulation assay prior and after Nvoc removal, which showed that neither the alanine 49 nor the attached Nvoc group impair the activity of the analog The study shows that lysine 56 is a potential site to introduce labels site-specifically in SDF-1 (C) 2010 Wiley Periodicals, Inc Biopolymers (Pept Sci) 94 771-778, 2010