Purification of surfactant protein A from dog lung by reconstitution with surfactant lipids.
We have developed a simple method for purification of surfactant major apoprotein (SP-A, MW 34-38 kD) from dog lungs with high yield and purity. Lipids and proteins of partially purified surfactant were dissociated by sodium deoxycholate (DOC, 100 mM, 37 degrees C, 30 min), diluted 1:10 with borate buffer containing 3 mM CaCl2, and dialysate in the same buffer to reconstitute the lipids and proteins (4 degrees C, 48 h). The reconstituent and the partially purified surfactant were purified by ultracentrifugation on a discontinuous sucrose density gradient. Protein was isolated from the reconstituent and from the purified surfactant by delipidation, and the yields and purities were assessed by one-dimensional SDS-PAGE and 2-dimensional electrophoresis (isoelectric focusing, SDS-PAGE). We found that the surface pressure-time adsorption isotherm, minimum surface tension, and the yield and composition of lipids of the reconstituent were identical with those from the purified surfactant. Only about 0.25% of the DOC used for dissociation remained with the reconstituent and it did not affect surface properties of the reconstituent. The yield of SP-A in the reconstituent was almost the same as that in the purified surfactant, but the former contained no plasma protein whereas the latter contained significant amounts. The amino acid composition and the partial N-terminal amino acid sequence of SP-A were the same as those from the purified surfactant. Reconstituent prepared from surfactant lipids and SP-A adsorbed more rapidly and reached a higher final surface pressure than did the surfactant lipids alone. These results demonstrate that large quantities of SP-A can be purified by reconstitution with surfactant lipids, and that the purified protein is biophysically active.
Liau
D F
DF
Department of Pathology, St. Luke's-Roosevelt Hospital Center, Columbia University College of Physicians and Surgeons, New York, NY 10025.
Ryan
S F
SF
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003840:Deoxycholic Acid; D004285:Dogs; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification; D007525:Isoelectric Focusing; D008055:Lipids / Q000032:analysis; D008168:Lung / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010743:Phospholipids / Q000032:analysis; D011510:Proteolipids / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology
D000596:Amino Acids; D006023:Glycoproteins; D008055:Lipids; D010743:Phospholipids; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D003840:Deoxycholic Acid