Biomedical subjects

Robert A Moxley

Publications and source records attributed to Robert A Moxley.

4 recordsLinked to original sources

PubMed2006

Promoter trapping of c-jun promoter-binding transcription factors.

A new method called promoter trapping was developed to purify promoter-protein complex using the c-jun promoter (-200+81) as a model, which was shown to have significant promoter activity. Polymerase chain reaction (PCR), lambda exonuclease digestion combined with (AC)(5)-Sepharose DNA affinity chromatography were used to produce c-jun promoter with a (GT)(5) tail at each 3' end. The intact promoter and different length pieces with one or two (GT)(5) tails had almost the same capacity to bind with (AC)(5)-Sepharose. In solution, tailed c-jun promoter (60 nM) and competitor poly dI:dC (30 ng/microl) was incubated with crude HEK293 nuclear extract to form a large protein-promoter complex, and the complex was then trapped by (AC)(5)-Sepharose by centrifugation or on a column. Compared with a popular alternative method, called here the immobilized promoter method, the products of promoter trapping were purer. The preinitiation complex purified by promoter trapping had the expected components including RNA polymerase II, TATA-box binding protein (TBP), TFIIF subunit RAP74, and transcription factor SP1, and transcribed RNA in vitro. Thus, the promoter trapping approach provides a useful tool for the purification and investigation of transcription complexes.

Blotting, Western↗

PubMed2005

Oligonucleotide trapping method for transcription factor purification systematic optimization using electrophoretic mobility shift assay.

Oligonucleotide trapping, where a transcription factor-DNA response element complex is formed in solution and then recovered (trapped) on a column, was optimized for the purification of CAAT/enhancer binding protein (C/EBP) from rat liver nuclear extract. Electrophoretic mobility shift assays (EMSAs) with ACEP24(GT)5 oligonucleotide, containing the CAAT element, was used to estimate thebinding affinity and concentration of C/EBP in the nuclear extract and then low concentrations of protein and oligonucleotide, which favor specific binding, were used for all further experiments. Also using EMSA, the highest concentrations of competitors, which inhibit non-specific binding but do not inhibit oligonucleotide binding by C/EBP, were determined to be 932 nM T18 (single-stranded DNA), 50 ng/ml heparin (non-DNA competitor), and 50 microg/ml poly(dI:dC) (duplex DNA). Inclusion of 0.1% Tween-20 improved DNA binding. For complex formation, 110 microg nuclear extract was diluted to 0.2 nM C/EBP (apparent Kd of C/EBP) and 1.34 nM ACEP24(GT)5 was added, along with Tween-20 and the competitors. After incubation, the complex was trapped by annealing the (GT)5 tail of the C/EBP-[ACEP24(GT)5] complex to an (AC)5-Sepharose column under flow at 4 degrees C. The column was washed with 0.4 M NaCl and the protein eluted with 1.2 M NaCl. The purification typically resulted in two proteins of apparent molecular mass 32000 and 38000. The smaller one, the major product, was identified to be C/EBP-alpha. The yield was 2.1 microg (66 pmol) of purified C/EBP-alpha p32. This systematic approach to oligonucleotide trapping is generally applicable for the purification of other transcription factors.

Base Sequence↗

PubMed2004

Effect of the detergent Tween-20 on the DNA affinity chromatography of Gal4, C/EBPalpha, and lac repressor with observations on column regeneration.

C/EBPalpha, Gal4, and lac repressor, representing three different transcription factor homology families, were expressed as fusion proteins and used to characterize the effects of column aging, Mg2+, the nonionic detergent Tween-20, column loading, and bovine serum albumin on DNA-affinity chromatography. When lac-repressor-beta-galactosidase fusion protein is loaded onto a new DNA-Sepharose column, less elutes from a new column than one that has been used two or more times. Higher amounts of lac repressor, the Green Fluorescent Protein fusions with CAAT enhancer binding protein (C/EBPalpha) and Gal4, elute from the columns when 0.1% Tween-20 is added to the mobile phase. The amount of improvement found depends upon the transcription factor studied and the amount of the protein loaded on the column; lac repressor and Gal4 are eluted in higher amounts over a large range of protein loads while C/EBP shows the greatest effect at low protein loads. This detergent effect is seen when either Sepharose or silica is used for the stationary phase. Including bovine serum albumin in the mobile phase gives a similar though lesser improvement to that observed with Tween-20. Mg2+ or EDTA in the mobile phase gave similar chromatography for C/EBP; since EDTA protects columns from DNases, its inclusion in the mobile phase is preferred. After extended use, the DNA affinity columns no longer bind transcription factors and this is not due to losses of DNA from the columns. Two simple methods (sodium dodecylsulfate and KSCN) were developed to regenerate such worn out columns.

Base Sequence↗

PubMed2003

Methods for transcription factor separation.

Recent advances in the separation of transcription factors (TFs) are reviewed in this article. An overview of the transcription factor families and their structure is discussed and a computer analysis of their sequences reveals that while they do not differ from other proteins in molecular mass or isoelectric pH, they do differ from other proteins in the abundance of certain amino acids. The chromatographic and electrophoretic methods which have been successfully used for purification and analysis are discussed and recent advances in stationary and mobile phase composition is discussed.

Base Sequence↗