Production in Escherichia coli of a recombinant C-terminal truncated precursor of surfactant protein B (rproSP-BΔc). Structure and interaction with lipid interfaces

SP-B, a protein absolutely required to maintain the lungs open after birth, is synthesized in the pneumocytes as a precursor containing C-terminal and N-terminal domains flanking the mature sequence. These flanking-domains are cleaved to produce mature SP-B, coupled with its assembly into pulmonary surfactant lipid–protein complexes. In the present work we have optimized over-expression in Escherichia coli and purification of rproSP-BΔC, a recombinant form of human proSP-B lacking the C-terminal flanking peptide, which is still competent to restore SP-B function in vivo. rProSP-BΔC has been solubilized, purified and refolded from bacterial inclusion bodies in amounts of about 4 mg per L of culture. Electrophoretic mobility, immunoreactivity, N-terminal sequencing and peptide fingerprinting all confirmed that the purified protein had the expected mass and sequence. Once refolded, the protein was soluble in aqueous buffers. Circular dichroism and fluorescence emission spectra of bacterial rproSP-BΔC indicated that the protein is properly folded, showing around 32% α-helix and a mainly hydrophobic environment of its tryptophan residues. Presence of zwitterionic or anionic phospholipids vesicles caused changes in the fluorescence emission properties of rproSP-BΔC that were indicative of lipid–protein interaction. The association of this SP-B precursor with membranes suggests an intrinsic amphipathic character of the protein, which spontaneously adsorbs at air–liquid interfaces either in the absence or in the presence of phospholipids. The analysis of the structure and properties of recombinant proSP-BΔC in surfactant-relevant environments will open new perspectives on the investigation of the mechanisms of lipid and protein assembly in surfactant complexes.