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.
Zaltash
S
S
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Johansson
J
J
D000818:Animals; D001665:Binding Sites; D004926:Escherichia coli; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D011487:Protein Conformation; D011498:Protein Precursors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011663:Pulmonary Surfactants / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D014357:Trypsin / Q000378:metabolism
D006023:Glycoproteins; C483345:PSAP protein, human; D011498:Protein Precursors; D011510:Proteolipids; D011663:Pulmonary Surfactants; D011993:Recombinant Fusion Proteins; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D014357:Trypsin