Aim: To test the hypothesis that precursor flanking sequences assist in the folding of mature Surfactant Protein B (SP-B).

Rationale: A 79 amino acid form of SP-B is one of the major apoproteins of pulmonary surfactant. Mutations in the SP-B gene resulting in SP-B deficiency lead to fatal respiratory disease in humans and genetically altered mice. SP-B is first translated as a preproprotein of 381 amino acids(preSP-B) which is then processed by the type II alveolar cell to the active homodimeric 79 amino acid surfactant apoprotein (SP-B). The sequences flanking SP-B in preSP-B each contain 6 cysteines with the same periodicity found in SP-B. Previous attempts to express SP-B have failed to achieve correct disulfide pairing and full protein activity.

Methods: As the proteases involved in releasing SP-B from preSP-B are not known the full length cDNA for human preSP-B was first mutated to insert hydroxylamine cleavage sites at residues 209 and 290. Modified preSP-B was expressed in E.coli and >90% purified by isolating inclusion granules. PreSP-B was denatured and reduced in a 6M urea-DTT buffer and then refolded in a non-denaturing buffer containing glutathione to assist in correct disulfide pairing. The soluble refolded product was purified by nickel affinity chromatography and characterized by size exclusion chromatography, SDS-PAGE, circular dichroism, determination of free SH content and hydroxylamine cleavage.

Results: PreSP-B was expressed as an insoluble product routed to inclusion granules. Following refolding the majority of preSP-B was soluble in physiologic buffer conditions. A significant amount of the refolded protein was monomeric (Mr 41 kDa) by size exclusion chromatography and non-reducing SDS-PAGE, the remainder was aggregated into variable higher MW forms. The monomeric fraction had the predicted number of S-S and free SH bonds and stable secondary structure (25% alpha helix, 13% anti parallel beta sheet, 25% turns, and 34% random coil).

Conclusions: These results suggest preSP-B can be expressed in E.coli and refolded into a stable monomer with the predicted secondary structure and disulfide assignment of the native SP-B precursor. This system should be useful for structure-function studies.