Expression, purification and characterization of the precursor of human pulmonary surfactant protein B (preproSPB) produced in Escherichia coli

Pulmonary surfactant protein B (SP-B) is involved in the transfer of phospholipid molecules from specific lipid/protein assemblies produced by pneumocytes to form surface active films at the air-liquid interface of lungs. The lack of SP-B is lethal, being its absence associated with an irreversible respiratory failure at birth. In vivo, SP-B is synthesized as a larger precursor, preproSP-B, of 381 amino acids, which Suffers proteolytic processing to remove N-terminal and C-terminal flanking propeptides during the exocytic pathway ending in the secretory lamellar bodies as the mature active 79-residue (9 kDa) polypeptide. Human preproSP-B has been cloned and expressed in strain BL21 (DE3) of E.coli and the recombinant His-tagged preproSP-B form of 42 kDa has been purified by affinity chromatography, After electrophoretic analysis and Western Blot detection, a preliminar structural characterization has been carried Out by different spectroscopic methods with the ultimate objective of analyzing how the protein behaves Under physiologically relevant en viron mental conditions. Production at high yield of preproSP-B, followed by a limited proteolysis will offer new possibilities in the development of the next generation of entirely synthetic therapeutic surfactants.