Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity.
Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich alpha helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new preparations for therapeutic use. We describe herein the recombinant production in bacterial cultures of SP-C variants containing phenylalanines instead of the palmitoylated cysteines of the native protein, as fusions to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two recombinant SP-C variants so obtained retained more than 50% alpha-helical content and showed surface activity comparable to the native protein, as measured by surface spreading of lipid/protein suspensions and from compression pi-A isotherms of lipid/protein films. Compared to the protein purified from porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations.
Lukovic
Dunja
D
Departament de Bioquímica i Biologia Molecular, Universitat de València, E-46 100 Burjassot (Valencia), Spain.
Plasencia
Inés
I
Taberner
Francisco J
FJ
Salgado
Jesús
J
Calvete
Juan J
JJ
Pérez-Gil
Jesús
J
Mingarro
Ismael
I
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D004337:Drug Carriers; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D037721:Pulmonary Surfactant-Associated Protein C; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013499:Surface Properties
D004337:Drug Carriers; D010455:Peptides; D010743:Phospholipids; D037721:Pulmonary Surfactant-Associated Protein C; D011994:Recombinant Proteins; C489679:SFTPC protein, human