Expression and Characterization of Saposin-Like Proteins
253
exhibits about 35 % a-helical structure, which is similar to the approximatly 45 %
helix of SP-B in dodecylphosphocholine micelles estimated by CD spectroscopy.
Limited proteolysis of rproSP-B occur predominantly between the three tandem
saposin-like domains that were 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 (9).
4
Expression of Recombinant NK-Lysin
We aim to express single saposin domains, especially NK-lysin. For this purpose
fragments corresponding to NK-lysin were PCR-amplified from porcine bone
marrow cDNA. Probes which correspond to the 5' and 3' ends ofNK-lysin (nucleotides 360-380 and 573-593 of porcine proNK-lysin) were used. The resulting
product was inserted into the pET15b vector, which gives an N-terminal poly-His
tag fused to the recombinant proteins, and sequenced by the dideoxy chaintermination method. The ligation product was transferred into E.coli JM 109 cells
for selection of plasmid-carrying clones. Finally, pET15b vector with inserted
NK-lysin DNA was transferred to E.coli BL21 DE3 cells, which contain an integrated copy of the T7 DNA polymerase gene under control of the lac UV5 promoter. The recombinant protein was purified from inclusion bodies by resolubilisation with 8 M urea and subsequent metal affinity chromatography, and
reduced/reoxidised by treatment with DTT followed by dialysis against DTT-free
buffer. The recombinant protein was subjected to SDS/PAGE which gives detectable bands at about 11 kDa, which is in agreement with the expected mass of
10.954 kDa of His-tag/NK-lysin fusion protein.
5
Discussion
ProSP-B fused to an N-terminal poly-His tag was expressed in E. coli and purified
from inclusion bodies by metal affinity chromatography after resolubilisation
with 2.5 % (w/v) SDS (9). This yields approximately 1.5 mg rproSP-B/L cell culture and the recombinant protein is concluded to be folded into a native-like conformation and to form disulphide-dependent oligomers. The present CD spectrum and limited proteolysis data lend experimental support to the suggestion
that proSP-B is composed of three tandem saposin-like domains. Of the three
saposin-like domains present in proSP-B, only SP-B has been isolated. It is therefore an open question whether the remaining two saposin-like domains in proSPB are processed to yield unique entities, or if the sole function of proSP-B is to
give rise to SP-B. Sequence alignments of canine, rabbit, rat and human proSP-B
show that the first and second (i.e. SP-B) domains exhibit a high degree of conservation, while the third domain is little conserved. Notably, the proSP-B Nterminal part, but not the C-terminal region, is required for its processing and
intracellular targeting (10,11). The conservation of the first saposin-like domain
may indicate that it is particularly important for proSP-B functions, or that it
plays a role also after processing of proSP-B to SP-B.
253
exhibits about 35 % a-helical structure, which is similar to the approximatly 45 %
helix of SP-B in dodecylphosphocholine micelles estimated by CD spectroscopy.
Limited proteolysis of rproSP-B occur predominantly between the three tandem
saposin-like domains that were 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 (9).
4
Expression of Recombinant NK-Lysin
We aim to express single saposin domains, especially NK-lysin. For this purpose
fragments corresponding to NK-lysin were PCR-amplified from porcine bone
marrow cDNA. Probes which correspond to the 5' and 3' ends ofNK-lysin (nucleotides 360-380 and 573-593 of porcine proNK-lysin) were used. The resulting
product was inserted into the pET15b vector, which gives an N-terminal poly-His
tag fused to the recombinant proteins, and sequenced by the dideoxy chaintermination method. The ligation product was transferred into E.coli JM 109 cells
for selection of plasmid-carrying clones. Finally, pET15b vector with inserted
NK-lysin DNA was transferred to E.coli BL21 DE3 cells, which contain an integrated copy of the T7 DNA polymerase gene under control of the lac UV5 promoter. The recombinant protein was purified from inclusion bodies by resolubilisation with 8 M urea and subsequent metal affinity chromatography, and
reduced/reoxidised by treatment with DTT followed by dialysis against DTT-free
buffer. The recombinant protein was subjected to SDS/PAGE which gives detectable bands at about 11 kDa, which is in agreement with the expected mass of
10.954 kDa of His-tag/NK-lysin fusion protein.
5
Discussion
ProSP-B fused to an N-terminal poly-His tag was expressed in E. coli and purified
from inclusion bodies by metal affinity chromatography after resolubilisation
with 2.5 % (w/v) SDS (9). This yields approximately 1.5 mg rproSP-B/L cell culture and the recombinant protein is concluded to be folded into a native-like conformation and to form disulphide-dependent oligomers. The present CD spectrum and limited proteolysis data lend experimental support to the suggestion
that proSP-B is composed of three tandem saposin-like domains. Of the three
saposin-like domains present in proSP-B, only SP-B has been isolated. It is therefore an open question whether the remaining two saposin-like domains in proSPB are processed to yield unique entities, or if the sole function of proSP-B is to
give rise to SP-B. Sequence alignments of canine, rabbit, rat and human proSP-B
show that the first and second (i.e. SP-B) domains exhibit a high degree of conservation, while the third domain is little conserved. Notably, the proSP-B Nterminal part, but not the C-terminal region, is required for its processing and
intracellular targeting (10,11). The conservation of the first saposin-like domain
may indicate that it is particularly important for proSP-B functions, or that it
plays a role also after processing of proSP-B to SP-B.