Identification by phage display of single-domain antibody fragments specific for the ODD domain in hypoxia-inducible factor 1alpha.
Hypoxia triggers the transcription of genes responsible for cell survival via the key player transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha). Overexpression of this protein has been implicated in cardiovascular disorders, carcinogenesis and cancer progression. For functional and diagnostic studies on the HIF-1alpha protein, we have identified single-domain antibody fragments directed against this protein by using a llama-derived nonimmune phage display library. This library displays the variable domains of the heavy-chain antibody subclass, found in these animals. Phage display selection with six recombinant HIF-1alpha proteins yielded five different antibody fragments. By epitope-mapping, we show that all five antibody fragments bind within the functionally important oxygen-dependent degradation domain of the HIF-1alpha protein. Two of these antibody fragments were engineered into bivalent antibodies that were able to detect human HIF-1alpha by immunohistochemistry, Western blotting and immunoprecipitation, and mouse HIF-1alpha by immunofluorescence and immunoprecipitation. These are the first single-domain antibody fragments that may be used in exploration of HIF-1alpha as a possible therapeutic target through molecular applications.
Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
Arjan J
AJ
Groot
Peter
P
Verheesen
Elike J
EJ
Westerlaken
Eelke H
EH
Gort
Petra
P
van der Groep
Niels
N
Bovenschen
Elsken
E
van der Wall
Paul J
PJ
van Diest
Avi
A
Shvarts
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D001667:Binding, Competitive; D002161:Camelids, New World; D003001:Cloning, Molecular; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000032:analysis / Q000737:chemistry / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000032:analysis* / Q000096:biosynthesis / Q000737:chemistry; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry
D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011994:Recombinant Proteins