Expression and purification of recombinant NFI proteins for functional analysis.
Nuclear factor I (NFI) is a transcription factor playing wide role in signal transduction pathways and developmental processes in higher eukaryotes. In order to produce recombinant NFI proteins for functional and structural studies, full length cDNAs of individual isoforms were subcloned into pETM30 vector and expressed in Escherichia coli. Although the fusion proteins containing both glutathione S-transferase (GST) and His6 tags at the N-terminus could be overexpressed in detectable amounts, they were found mainly, if not exclusively, in insoluble form. Purification yield was improved by modification of cell disruption procedure and by the use of detergent Tween 20. The final purification strategy represents a triple-helix affinity chromatography consisting of prepurification of bacterial lysate on Heparin-Sepharose with subsequent immobilized metal affinity and glutathione affinity chromatography. Heparin chromatography was crucial for obtaining active NFI proteins, whereas the other steps significantly improved the purity of isolated proteins. As demonstrated by EMSA and DNase I protection assay, the recombinant proteins were able to recognize their cognate DNA sequences.
Kollárovic
Gabriel
G
Laboratory of Molecular Biology, Cancer Research Institute, Slovak Academy of Sciences, Vlárska 7, 833 91 Bratislava, Slovakia. gabriel.kollarovic@savba.sk
Majera
Dusana
D
Luciaková
Katarína
K
Baráth
Peter
P
D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D007118:Immunoassay; D047468:Immunoprecipitation; D051839:NFI Transcription Factors / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D000906:Antibodies; D002457:Cell Extracts; D051839:NFI Transcription Factors; D020033:Protein Isoforms; D011994:Recombinant Proteins; D004247:DNA