252
S. ZALTASH and J. JOHANSSON
Table 18.1. The saposin family. SP-B, amoebapores (which are pore-forming polypeptides from Entamoeba hisytolytica), parts of acid sphingomyelinase, plant aspartic protease and acyloxyacylhydrolase, the saposins (which promote enzymatic degradation of sphingolipids in lysosomes) and NKlysin (which is an antibacterial and tumourolytic polypeptide from nature killer cells), exhibit 17 to
24 % pairwise residue identities
* NK-lysin
* SP-B
* Amoebapores
* Sapos ins
* Parts of acyloxyacylhydrolase
* Parts of acid sphingomyelinase
* Parts of plant aspartic protease
ond of these repeats (residues 201-279 in proSP-B). Moreover, the intramolecular
disulphide patterns in SP-B, NK-Iysin (which is an antibacterial and tumourolytic
polypeptide from Natural Killer cells), and saposins Band C are identical (2, 6,
7). We have expressed human proSP-B in E. coli, and characterized the recombinant protein in terms of overall secondary structure and susceptibility to limited proteolysis with trypsin. This shows that proSP-B exhibits about 35 %
a-helical structure and is preferentially cleaved between the three proposed
saposin-like domains. Currently we aim to express single saposin domains, especially NK-Iysin, for studies of structural and functional properties.
3
Analysis of Recombinant proSP-B
We have analyzed the domain organization of recombinant proSP-B. The cDNA
coding for the precusor of human SP-B has been cloned and sequenced and codes
for a protein of 381 amino acid residues (8). The 381-residue human proSP-B
fused to an N-terminal poly-His tag was expressed in E. coli. The recombinant
protein was purified from inclusion bodies by resolubilisation with 2.5 % (w/v)
SDS and subsequent metal affinity chromatography after removal of SDS by dialysis (Fig. 18.I). Recombinant proSP-B solubilised in sodium phosphate buffer
Purification of Recombinant Poly-His-proSP-B
1. IPTG-induced E. coli BL21 cells harvested.
2. Bacteria resupended in 20 mM Tris-HCI, 100 mM NaCI, pH 8.0 (buffer A).
3. Sonication 10 times, 60 Hz, 30 secs with an interval of 30 sees.
4. Centrifugation at 15000 rpm, 15 mins.
5. Pellet resuspended in buffer A containing 2.5 % SDS.
6. Sonication and incubation at 37°C, 30 mins.
7. Centrifugation 15000 rpm, IS mins. Supernatant divided into two aliquots.
t
t
+SDS
-SDS
t
t
8. Dialysis against 20 mM Tris-HCl, pH 8.0 in the presence or absence of 2.5 % SDS, respectively.
9. Metal affinity chromatography. Elution with 100 mM imidazole.
Fig. 18.1. Purification strategy for rproSP B. The stategy for purification of rproSP-B is outlined
S. ZALTASH and J. JOHANSSON
Table 18.1. The saposin family. SP-B, amoebapores (which are pore-forming polypeptides from Entamoeba hisytolytica), parts of acid sphingomyelinase, plant aspartic protease and acyloxyacylhydrolase, the saposins (which promote enzymatic degradation of sphingolipids in lysosomes) and NKlysin (which is an antibacterial and tumourolytic polypeptide from nature killer cells), exhibit 17 to
24 % pairwise residue identities
* NK-lysin
* SP-B
* Amoebapores
* Sapos ins
* Parts of acyloxyacylhydrolase
* Parts of acid sphingomyelinase
* Parts of plant aspartic protease
ond of these repeats (residues 201-279 in proSP-B). Moreover, the intramolecular
disulphide patterns in SP-B, NK-Iysin (which is an antibacterial and tumourolytic
polypeptide from Natural Killer cells), and saposins Band C are identical (2, 6,
7). We have expressed human proSP-B in E. coli, and characterized the recombinant protein in terms of overall secondary structure and susceptibility to limited proteolysis with trypsin. This shows that proSP-B exhibits about 35 %
a-helical structure and is preferentially cleaved between the three proposed
saposin-like domains. Currently we aim to express single saposin domains, especially NK-Iysin, for studies of structural and functional properties.
3
Analysis of Recombinant proSP-B
We have analyzed the domain organization of recombinant proSP-B. The cDNA
coding for the precusor of human SP-B has been cloned and sequenced and codes
for a protein of 381 amino acid residues (8). The 381-residue human proSP-B
fused to an N-terminal poly-His tag was expressed in E. coli. The recombinant
protein was purified from inclusion bodies by resolubilisation with 2.5 % (w/v)
SDS and subsequent metal affinity chromatography after removal of SDS by dialysis (Fig. 18.I). Recombinant proSP-B solubilised in sodium phosphate buffer
Purification of Recombinant Poly-His-proSP-B
1. IPTG-induced E. coli BL21 cells harvested.
2. Bacteria resupended in 20 mM Tris-HCI, 100 mM NaCI, pH 8.0 (buffer A).
3. Sonication 10 times, 60 Hz, 30 secs with an interval of 30 sees.
4. Centrifugation at 15000 rpm, 15 mins.
5. Pellet resuspended in buffer A containing 2.5 % SDS.
6. Sonication and incubation at 37°C, 30 mins.
7. Centrifugation 15000 rpm, IS mins. Supernatant divided into two aliquots.
t
t
+SDS
-SDS
t
t
8. Dialysis against 20 mM Tris-HCl, pH 8.0 in the presence or absence of 2.5 % SDS, respectively.
9. Metal affinity chromatography. Elution with 100 mM imidazole.
Fig. 18.1. Purification strategy for rproSP B. The stategy for purification of rproSP-B is outlined