Secondary structure and limited proteolysis give experimental evidence that the precursor of pulmonary surfactant protein B contains three saposin-like domains.

The 42 kDa precursor of surfactant protein B generates the 79 residue mature SP-B polypeptide, which belongs to the family of saposin-like proteins and has unique functional roles in pulmonary surfactant. From sequence comparisons it has been suggested that proSP-B, in addition to SP-B, contains two saposin-like domains, but their existence has until now not been experimentally verified. The 381 residue human proSP-B was now fused to an N-terminal poly-His tag, expressed in Escherichia coli, and purified from inclusion bodies by resolubilisation with 2.5% (w/v) SDS and, after removal of SDS, subsequent metal affinity chromatography. Recombinant proSP-B thus obtained exhibits about 35% alpha-helical structure in sodium phosphate buffer and is proteolytically cleaved preferentially between the three saposin-like domains. These results experimentally support that proSP contains, in addition to SP-B, two further saposin-like domains.