Expression of a synthetic gene encoding the Asp-hemolysin from Aspergillus fumigatus in Escherichia coli.
Asp-hemolysin is a hemolytic toxin from Aspergillus fumigatus and is a specific binding protein with high affinity for oxidized low density lipoprotein (Ox-LDL). As a first step in clarifying the structure-function relationship of Asp-hemolysin, we expressed Asp-hemolysin in Escherichia coli (E. coli) as a fusion protein with a maltose-binding protein (MBP) and purified it by affinity chromatography on an amylose resin. The apparent molecular size of the protein produced by E. coli was approximately 57 kDa, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This is consistent with the predicted molecular size of 56.9 kDa for a fusion protein which includes 14.2 kDa of Asp-hemolysin and 42.7 kDa from MBP. The purified recombinant Asp-hemolysin showed an immunoreactivity with the anti-Asp-hemolysin antibody as revealed by western blot analysis. Furthermore, in dot blot analysis, MBP-Asp-hemolysin fusion protein exhibited binding activity to Ox-LDL as did native Asp-hemolysin.
Kumagai
Takeshi
T
The First Department of Hygienic Chemistry, Tohoku Pharmaceutical University, Sendai, Japan. ta-kuma@tohoku-pharm.ac.jp
Kudo
Yoichi
Y
Fukuchi
Yuji
Y
Ebina
Keiichi
K
Yokota
Katsushi
K
D018528:ATP-Binding Cassette Transporters; D001232:Aspergillus fumigatus / Q000235:genetics*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D008077:Lipoproteins, LDL / Q000378:metabolism; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010084:Oxidation-Reduction; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; C089497:Asp-HS protein, Aspergillus fumigatus; D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D006460:Hemolysin Proteins; D008077:Lipoproteins, LDL; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; C461599:maltose transport system, E coli