ABSTRACT
The focus of this review is on DNA affinity chromatography, which is the most powerful tool for purification of DNA binding proteins. The use of nonspecific-, sequence specific- and single stranded-DNA affinity columns in purification of various DNA binding proteins is discussed. The purification strategies for transcription factors, restriction enzymes, telomerases, DNA and RNA polymerase and DNA binding antibodies are described. Different applications of DNA affinity chromatography are presented.
Subject(s)
Chromatography, Affinity/methods , DNA-Binding Proteins/isolation & purification , Animals , Base Sequence , DNA/chemistry , DNA Primers , DNA-Binding Proteins/chemistry , DNA-Directed DNA Polymerase/isolation & purification , DNA-Directed RNA Polymerases/isolation & purification , Telomerase/isolation & purification , Templates, Genetic , Transcription Factors/isolation & purificationABSTRACT
Concatemers, tandem copies of DNA elements ligated together, are widely used for the DNA affinity chromatography of transcription factors. Purification of different transcription factors using discrete, concatemeric and T18:A18 tailed DNA affinity columns was studied. Columns having a discrete DNA sequence bound by cytidylic-adenylic-adenylic-thymidylic oligonucleotide (CAAT) enhancer binding protein (C/EBP) yields significantly more green fluorescent protein-C/EBP (GFP-C/EBP) fusion protein than a concatemeric DNA column made from five tandem repeats of the same DNA sequence. For lac repressor protein, the concatemeric and T18:A18 tailed columns show greater retention times than a discrete, untailed DNA affinity column. It was observed that the T18:A18 tailed column gives better resolution than either the discrete or concatemeric columns, of mixtures containing both lac repressor and GFP-C/EBP. Discrete concatemeric and T18:A18 tail columns all bound the Sp1 transcription factor and showed similar retention. The T18:A18 tailed column gives higher yield for Sp1 than the other columns. Our study shows concatemeric columns do not have any distinct advantage for the three different transcription factors we studied including Sp1, the original justification for the concatemeric approach.
Subject(s)
Chromatography, Affinity/methods , DNA/chemistry , Sepharose/chemistry , Transcription Factors/isolation & purification , Base Sequence , CCAAT-Enhancer-Binding Proteins/chemistry , Electrophoresis, Polyacrylamide Gel , Green Fluorescent Proteins , Luminescent Proteins/chemistry , Recombinant Fusion Proteins/analysisABSTRACT
A novel bi-column method for purifying transcription factors, using two different columns and two different elution strategies is described. Lac repressor elutes at lower heparin concentrations from a lower affinity lactose operatorl (Op1)-Sepharose column than from a higher affinity column containing the same sequence with a T18:A18 tail (Op1T18). A bi-column method was developed in which lac repressor fusion protein is eluted from the Op1-Sepharose with a low heparin concentration and trapped on a Op1T18-Sepharose column because of its higher affinity for the lac repressor protein. Elution of the latter column with buffer containing a high salt concentration gives significantly purer transcription factor than the conventionally used single column methods and removes residual heparin. Highly pure CAAT enhancer binding protein and the B3 transcription factor are also obtained by using variants of this bi-column method.
Subject(s)
Chromatography, Affinity/methods , Transcription Factors/isolation & purificationABSTRACT
The length of a DNA sequence attached to an affinity chromatography column affects column retention of transcription factors. Even when unrelated sequences such as a poly(A):poly(T) tail are included in a DNA sequence, transcription factors such as the lac repressor are bound more tightly by the column. The position of the additional sequences is also important. To compare coupling procedures, an identical DNA sequence was covalently attached to Sepharose by chemical coupling or produced enzymatically by template driven enzymatic primer extension. These two types of supports, containing the O1 operator sequence bound by lac repressor, were packed into identical columns and compared by purification of a lac repressor-beta-galactosidase fusion protein. We found that the purity and yield of proteins eluted from the two columns were similar. Overall, the results suggest that there is no significant advantage to either type of support for the purification of some proteins. The study revealed a potentially important effect of the length of DNA sequences on column selectivity.
Subject(s)
Bacterial Proteins/isolation & purification , Chromatography, Agarose/methods , DNA/chemistry , Escherichia coli Proteins , Repressor Proteins/isolation & purification , Chromatography, Affinity/methods , Lac Repressors , Recombinant Fusion Proteins/isolation & purificationABSTRACT
A novel method using heparin for eluting transcription factors from DNA-Sepharose columns was characterized. CAAT enhancer binding protein (C/EBP) or lac repressor fusion proteins were both eluted with heparin from columns containing specific DNA sequences coupled to cyanogen bromide activated Sepharose. The amount of the lac repressor chimera which eluted from the column was shown to increase with increases in the mobile phase heparin concentration. The elution of the protein was also shown to be dependent on the amount of DNA coupled to the column and more protein eluted from columns containing lesser amounts of DNA. These data suggest that heparin and DNA compete for binding to the protein; this competition causes elution. Comparison of heparin- and salt-eluted protein demonstrated the heparin-eluted fraction was significantly purer and comparable to that obtained by elution with isopropyl beta-D-thiogalactopyranoside, a lactose analog. Heparin elution represents an important new tool in the purification of transcription factors and other DNA-binding proteins by DNA affinity chromatography.
