CHAPTER 18
Expression and Characterization
of Saposin-Like Proteins
S. ZALTASH' and J. JOHANSSON'
1
Abstract
The 79 residue mature surfactant protein B is formed by proteolytic cleavage
from a larger precursor. SP-B belongs to the family of saposin-like proteins and
has unique functional roles in pulmonary surfactant. The 381-residue human
proSP-B fused to an N-terminal poly-His tag was expressed in E. coli, and purified from inclusion bodies by resolubilisation with 2.5 % (w/v) SDS followed by
metal affinity chromatography after removal of SDS. Recombinant proSP-B solubilised in sodium phosphate buffer exhibits about 35 % a-helical structure and is
preferentially proteolytically cleaved between the three tandem saposin-like
domains that have been proposed from amino acid sequence comparisons. These
results give experimental support to the possibility that proSP-B contains, in
addition to SP-B, two further saposin-like domains.
2
Introduction
Lung surfactant is a complex mixture of phospholipids and proteins. The main
function of this system is to reduce the surface tension at the alveolar airlliquid
interface. Four different surfactant-associated proteins have been purified. The
larger surfactant proteins A (SP-A) and D (SP-D), are hydrophilic, while SP-B and
sp-c are small and insoluble in water, see (1). SP-B and SP-C probably have
unique functional roles in the formation of the surface-active monolayer. These
two hydrophobic proteins are unrelated in structure, but both of the mature proteins are formed by proteolytic cleavage from larger precursors. The mature SP-B
polypeptide chain consists of 79 residues, forms disulphide-dependent homodimers, and displays about 45 % a-helical secondary structure in phospholipid
bilayers (2, 3). SP-B belongs to the family of saposin-like proteins (Table. 18.1).
SP-B and the other members of the saposin-like family exhibit 17-24 % pairwise
residue identities (4). From sequence alignments with saposins, the 42 kDa precursor of surfactant protein B (proSP-B) has been proposed to contain three tandem repeats of about 90 residues (5), where mature SP-B corresponds to the secI Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-l71 77 Stockholm,
Sweden.
Expression and Characterization
of Saposin-Like Proteins
S. ZALTASH' and J. JOHANSSON'
1
Abstract
The 79 residue mature surfactant protein B is formed by proteolytic cleavage
from a larger precursor. SP-B belongs to the family of saposin-like proteins and
has unique functional roles in pulmonary surfactant. The 381-residue human
proSP-B fused to an N-terminal poly-His tag was expressed in E. coli, and purified from inclusion bodies by resolubilisation with 2.5 % (w/v) SDS followed by
metal affinity chromatography after removal of SDS. Recombinant proSP-B solubilised in sodium phosphate buffer exhibits about 35 % a-helical structure and is
preferentially proteolytically cleaved between the three tandem saposin-like
domains that have been proposed from amino acid sequence comparisons. These
results give experimental support to the possibility that proSP-B contains, in
addition to SP-B, two further saposin-like domains.
2
Introduction
Lung surfactant is a complex mixture of phospholipids and proteins. The main
function of this system is to reduce the surface tension at the alveolar airlliquid
interface. Four different surfactant-associated proteins have been purified. The
larger surfactant proteins A (SP-A) and D (SP-D), are hydrophilic, while SP-B and
sp-c are small and insoluble in water, see (1). SP-B and SP-C probably have
unique functional roles in the formation of the surface-active monolayer. These
two hydrophobic proteins are unrelated in structure, but both of the mature proteins are formed by proteolytic cleavage from larger precursors. The mature SP-B
polypeptide chain consists of 79 residues, forms disulphide-dependent homodimers, and displays about 45 % a-helical secondary structure in phospholipid
bilayers (2, 3). SP-B belongs to the family of saposin-like proteins (Table. 18.1).
SP-B and the other members of the saposin-like family exhibit 17-24 % pairwise
residue identities (4). From sequence alignments with saposins, the 42 kDa precursor of surfactant protein B (proSP-B) has been proposed to contain three tandem repeats of about 90 residues (5), where mature SP-B corresponds to the secI Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-l71 77 Stockholm,
Sweden.