David E. Lyons

Purification and characterization of soluble forms of human and rat stem cell factor recombinantly expressed by Escherichia coli and by Chinese hamster ovary cells

Stem cell factor (SCF) is a novel, early-acting hematopoietic factor. It was isolated from the medium of a rat cell line in a soluble, processed form (Zsebo et al., 1990, Cell 63, 195). The cloned human and rat genes encode the soluble form plus additional C-terminal amino acids including a hydrophobic transmembrane domain (Martin et al., 1990, Cell 63, 203). We have recombinantly expressed forms of human and rat SCF corresponding to the soluble, processed form in Escherichia coli and in Chinese hamster ovary (CHO) cells. After expression in E. coli, folding and oxidation of the SCF polypeptides are required. The SCFs expressed in CHO cells are secreted into the medium in active state and, like the natural SCF, are glycosylated. Purification of the recombinant SCFs is described. Biological and biochemical characterization includes activity toward responsive human and mouse cell lines, N-terminal amino acid sequences, disulfide bond linkages, and sites of glycosylation.

Disulfide characterization of CD31 (PECAM)

Publisher Summary This chapter describes the disulfide characterization of CD31 (PECAM). CD31 is a 100 kDa integral membrane glycoprotein found in endothelial intercellular junctions, platelets, and monocytes. CD31 is believed to be involved in the regulation of endothelial cell migration, thus it is probably involved in vascular development, wound repair, and angiogenesis. Although binding of CD31 is homophilic, there is some evidence that it may bind other members of the cellular adhesion molecule family. Common to the adhesion molecules is the immunoglobulin (Ig) homology unit that consists of two β-sheets stabilized by a disulfide bond. Few adhesion molecule disulfide structures are determined in the chapter. Most disulfide structures are assumed by structural homology with other proteins in the same family or superfamily. The adhesion molecules are relatively, hydrophobic, and heavily glycosylated. Deglycosylation of the native protein ( N - and O - sites) reduce the complexity of peptide maps and simplify mass spectral data, allowing for simpler disulfide assignments.