Expression of biologically active C3a as fusion proteins.
We selected three kinds of plasmids for expression of C3a as fusion proteins. The proteins were purified by affinity chromatography using the respective specific resins, and their activities were measured by guinea pig platelet aggregation. We showed that polyhistidine (polyHis)-C3a fusion protein was able to exhibit 30% of the activity of natural C3a. However, glutathione S-transferase (GST)-C3a fusion protein exhibited only 10% of such activity, and no activity was measured for maltose binding protein (MBP)-C3a fusion protein. The purified polyHis-C3a fusion protein was attached to the Ni-NTA agarose column in an attempt to isolate the C3a receptor from guinea pig platelets. The C3a binding protein isolated from digitonin-solubilized guinea pig platelet membrane was approximately 50 kDa on SDS-polyacrylamide gel. This is the first report of C3a fusion protein production with biological activity.
Fukuoka
Y
Y
Department of Molecular Immunology, Tohoku University, Sendai, Japan.
Tachibana
T
T
Yasui
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001792:Blood Platelets / Q000276:immunology; D002352:Carrier Proteins; D002846:Chromatography, Affinity; D015926:Complement C3a / Q000096:biosynthesis* / Q000302:isolation & purification / Q000502:physiology; D005982:Glutathione Transferase; D006168:Guinea Pigs; D006639:Histidine; D015151:Immunoblotting; D066298:In Vitro Techniques; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D010455:Peptides; D010974:Platelet Aggregation; D011951:Receptors, Complement / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D010455:Peptides; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; C085567:complement C3a receptor; C033223:polyhistidine; D006639:Histidine; D015926:Complement C3a; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase