Production of surfactant protein C in the baculovirus expression system: the information required for correct folding and palmitoylation of SP-C is contained within the mature sequence.
Surfactant protein C (SP-C) is synthesized in the alveolar type II cells of the lung as a 21 kDa propeptide which is proteolytically processed to a 4.2 kDa mature active form. The main function of this extremely hydrophobic protein is to enhance lipid insertion into the air/liquid interface in the lung upon inhalation. This is necessary to maintain a relatively low surface tension at this interface during breathing. In this report we describe the production of mature human SP-C in the baculovirus expression system. The recombinant protein contains a secondary structure with a high alpha-helical content (73%), comparable to native SP-C, as determined by circular dichroism and attenuated total reflection Fourier transform infrared analysis. The expressed protein is a mixture of dipalmitoylated (15%) and non-palmitoylated SP-C. This suggests that the information required for palmitoylation is contained within the sequence of the mature protein. The activity of the protein to insert phospholipids into a preformed monolayer of lipids at an air/liquid interface was determined with a captive bubble surfactometer. Recombinant SP-C significantly reduced the surface tension at the air/liquid interface during dynamic expansion and compression. We conclude that correctly folded, dipalmitoylated and active SP-C can be expressed in the baculovirus expression system. Our results may facilitate investigations into the relation between structure and function of SP-C and into protein palmitoylation in general.
Veldhuizen
E J
EJ
Laboratory of Veterinary Biochemistry and Institute of Biomembranes, Utrecht University, 3508 TD, Utrecht, The Netherlands.
Batenburg
J J
JJ
Vandenbussche
G
G
Putz
G
G
van Golde
L M
LM
Haagsman
H P
HP
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D015870:Gene Expression; D007313:Insecta; D013058:Mass Spectrometry; D019308:Palmitic Acid / Q000737:chemistry; D017510:Protein Folding; D011510:Proteolipids / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011663:Pulmonary Surfactants / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared
D011510:Proteolipids; D011663:Pulmonary Surfactants; D019308:Palmitic Acid