Event Title
Cloning and Expression of HIF 1a from the gulf killifish Fundulus heteroclitus
Faculty Sponsor
Bernard Rees
Submission Type
Poster
Description
The hypoxia-inducible factor (HIF) is a transcription factor that regulates gene expression in animals exposed to low oxygen concentrations, or hypoxia. Previously, we have generated polyclonal antibodies against HIF-1α from the killifish Fundulus heteroclitus. The purpose of this research is to clone and express a fragment of HIF-1α to use in affinity purification of these antibodies. A ~300 bp region of F. heteroclitus HIF-1α cDNA was cloned into the Pet-Duet vector and transformed into E. coli (DH5α) cells. After sequence confirmation, the HIF-1α Pet-Duet plasmid was transformed into E. coli Bl21 (DE3) cells, which were induced to express recombinant HIF-1 α protein fragment. The protein fragment was partially purified by Ni-NTA agarose affinity chromatography. This resulted in a protein of the predicted molecular weight (~13 kDa), was seen in Coomassie-stained gels and by western blotting with the HIF-1α antibodies. However, several other proteins co-purify with the HIF-1 α fragment. Size exclusion chromatography was utilized in an attempt to further purify HIF-1 α. Use of multiple size exclusion media under differing conditions (native and denaturing) did not result in significant enrichment of the HIF-1 α fragment. Current efforts employ different strategies to obtain pure recombinant HIF-1 α protein fragment. /
Cloning and Expression of HIF 1a from the gulf killifish Fundulus heteroclitus
The hypoxia-inducible factor (HIF) is a transcription factor that regulates gene expression in animals exposed to low oxygen concentrations, or hypoxia. Previously, we have generated polyclonal antibodies against HIF-1α from the killifish Fundulus heteroclitus. The purpose of this research is to clone and express a fragment of HIF-1α to use in affinity purification of these antibodies. A ~300 bp region of F. heteroclitus HIF-1α cDNA was cloned into the Pet-Duet vector and transformed into E. coli (DH5α) cells. After sequence confirmation, the HIF-1α Pet-Duet plasmid was transformed into E. coli Bl21 (DE3) cells, which were induced to express recombinant HIF-1 α protein fragment. The protein fragment was partially purified by Ni-NTA agarose affinity chromatography. This resulted in a protein of the predicted molecular weight (~13 kDa), was seen in Coomassie-stained gels and by western blotting with the HIF-1α antibodies. However, several other proteins co-purify with the HIF-1 α fragment. Size exclusion chromatography was utilized in an attempt to further purify HIF-1 α. Use of multiple size exclusion media under differing conditions (native and denaturing) did not result in significant enrichment of the HIF-1 α fragment. Current efforts employ different strategies to obtain pure recombinant HIF-1 α protein fragment. /