Purification of nuclear factor I by DNA recognition site affinity chromatography

Philip J Rosenfeld, T. J. Kelly

Research output: Contribution to journalArticle

148 Citations (Scopus)

Abstract

Nuclear factor I (NF-I) is a cellular protein that enhances the initiation of adenovirus DNA replication in vitro. The enhancement of initiation correlates with the ability of NF-I to bind a specific nucleotide sequence within the viral origin of replication. We have developed a method for the purification of NF-I which is based upon the high affinity interaction between the protein and its recognition site. This approach may be generally applicable to the purification of other site-specific DNA binding proteins. The essential feature of the method is a two-step column chromatographic procedure in which proteins are first fractionated on an affinity matrix consisting of nonspecific (Escherichia coli) DNA and then on a matrix that is highly enriched in the specific DNA sequence that is recognized by NF-1. During the first step NF-I coeuutes with proteins that have similar general affinity for DNA. During the second step NF-I elutes at a much higher ionic strength than the contaminating nonspecific DNA binding proteins. The DNA recognition site affinity matrix used in the second step is prepared from a plasmid (pKB67-88) that contains 88 copies of the NF-I binding site. This plasmid was constructed by means of a novel cloning strategy that generates concatenated NF-I binding sites arranged exclusively in a direct head to tail configuration. Using this purification scheme, we have obtained a 2400-fold purification of NF-I from crude HeLa nuclear extract with a 57% recovery of specific DNA binding activity. Throughout the purification procedure NF-I retained the ability to enhance the efficiency of initiation of adenovirus DNA replication in vitro. Electrophoresis of the purified fraction on sodium dodecyl sulfate-polyacrylamide gels revealed a population of related polypeptides that ranged in apparent molecular weight from 66,000 to 52,000. The native molecular weight of NF-I deduced from gel filtration and glycerol sedimentation studies is 55,000 and the fractional ratio is 1.3. These results suggest that NF-I exists as a globular monomer in solution.

Original languageEnglish
Pages (from-to)1398-1408
Number of pages11
JournalJournal of Biological Chemistry
Volume261
Issue number3
StatePublished - Jan 1 1986
Externally publishedYes

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NFI Transcription Factors
Affinity chromatography
Affinity Chromatography
Purification
DNA
DNA-Binding Proteins
DNA Replication
Adenoviridae
Proteins
Plasmids
Molecular Weight
Molecular weight
Binding Sites
Replication Origin
Cloning
DNA sequences
Ionic strength
Electrophoresis
Sedimentation
Sodium Dodecyl Sulfate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification of nuclear factor I by DNA recognition site affinity chromatography. / Rosenfeld, Philip J; Kelly, T. J.

In: Journal of Biological Chemistry, Vol. 261, No. 3, 01.01.1986, p. 1398-1408.

Research output: Contribution to journalArticle

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