A selection system to study C5a-C5a-receptor interactions: phage display of a novel C5a anaphylatoxin, Fos-C5aAla27.
Binding and effector domains of the human anaphylatoxin C5a have been determined by either site directed mutagenesis or synthetic peptide studies. However, the lack of specific selection methods, which allow direct investigation of C5a-C5a-receptor interaction made these studies laborious. To overcome these limitations we have constructed a novel Fos-C5a expressed on the tip of a filamentous phage. To guarantee for a free C-terminus which is required for C5a activity C5a cDNA was cloned into the phagemid vector pJuFo. Helper phage infection of pJuFc-C5a transformed cells resulted in a mutant phage displaying Fos-C5a on its surface. However studies with Bt2cAMP differentiated U937 cells revealed that phage displayed Fos-C5a is functional inactive. Subsequently we replaced a nonconserved cysteine residue at position 27 by alanine and obtained Fos-C5aAla27. Both the purified and the phage displayed Fos-C5aAla27 proteins were functional active and induced enzyme release from differentiated U937 cells. In addition, purified Fos-C5aAla27 exhibited the same binding profile as compared to rhC5a. Fos-C5aAla27 displaying phages were mixed with phage harboring only the pJuFo plasmid at a ratio of 10(6). After four successive rounds of panning on differentiated U937 cells Fos-C5aAla27 phages were enriched to 100% as shown by C5a-specific ELISA. We expect this approach to prove helpful for studying C5a-C5a-receptor interactions. i.e. to screen C5a libraries for high affinity binders with agonistic or antagonistic properties directly on cells.
Hennecke
M
M
Institute of Medical Microbiology, Medical School Hannover, Germany.
Kola
A
A
Baensch
M
M
Wrede
A
A
Klos
A
A
Bautsch
W
W
Köhl
J
J
D000409:Alanine / Q000378:metabolism; D015703:Antigens, CD / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001435:Bacteriophages / Q000235:genetics*; D001667:Binding, Competitive; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D015936:Complement C5a / Q000378:metabolism*; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D009154:Mutation; D019151:Peptide Library; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D015703:Antigens, CD; D018076:DNA, Complementary; D019151:Peptide Library; D016760:Proto-Oncogene Proteins c-fos; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; D015936:Complement C5a; D000409:Alanine