Isolation and Characterization of Recombinant SP-B and SP-A

Recombinant human SP-B^N (MW, 8 KDa) was expressed in Escherichia coli BL21 (DE3) and purified over a Ni-NTA agarose column (Novagen) as previously described (5 (link)). Human surfactant protein A was isolated from bronchoalveolar lavage (BAL) of patients with alveolar proteinosis using a sequential butanol and octylglucoside extraction (8 (link)-10 (link)). The purity of SP-A and SP-B^N was verified by 1-dimensional SDS-PAGE in 12% acrylamide under reducing conditions. In addition, human SP-A was characterized by intrinsic fluorescence spectroscopy (8 (link)) and dynamic light scattering (DLS) (9 (link)). The oligomerization state of SP-A was assessed by electrophoresis under nondenaturing conditions (8 (link), 10 (link)), electron microscopy (8 (link)), and analytical ultracentrifugation as reported elsewhere (10 (link)). SP-A consisted of supratrimeric oligomers of at least 18 subunits (MW, 650 KDa). Biotinylated SP-A and SP-B^N were prepared as previously described (9 (link)). The structure and functional activity of biotinylated proteins were similar to those of unlabeled SP-A and SP-B^N. The endotoxin level of each protein was measured by the limulus amebocyte lysate endotoxin assay kit according to the manufacturer’s instructions (GenScript, USA). Endotoxin levels of the proteins were less than 0.15 EU/ml.