Semiquantitative Comparison of the DNA-Binding Activity of In Vitro-Synthesized Proteins

Structure-activity analysis of proteins requires the design of several mutant proteins. The expression, purification and accurate comparison of the activity of these different proteins can therefore be very tedious. The use of in vitro-translated proteins is a much faster way to obtain proteins suitable for activity comparison. Moreover, the conditions for in vitro translation are more similar to a eukaryotic environment than to a bacterial system from which proteins are often purified. Nevertheless, the direct comparison of the activity of the in vitro-translated proteins might not reflect a difference in their activity but rather a difference in the amount of protein synthesized in the extract, a problem that has not been addressed so far. We describe a rapid method that permits a semiquantitative comparison of the DNA-binding activity of in vitro-translated proteins. This method is based on the normalization of the DNA-binding activity to the amount of protein synthesized in the assay, permitting a more accurate estimation of the relative affinity for DNA of DNA-binding proteins. p53 was chosen as an example for a DNA-binding protein. The human p53 gene was cloned at the NcoI/BamHI sites of a pCITE-2a(+) vector (Novagen, Madison, WI, USA). The p53 plasmid was purified with a Wizard Plus Minipreps DNA Purification System (Promega, Madison, WI, USA) and ethanol-precipitated. The plasmid was resuspended in nuclease-free water and its concentration determined by spectrophotometry at 260 nm with a GeneQuant II apparatus (Pharmacia Biotech, Uppsala, Sweden). The p53 protein was in vitro-synthesized in rabbit reticulocyte lysate in the presence of 10 μCi of 35S-methionine (Amersham International plc, Little Chalfont, Bucks, England, UK) in a final volume of 25 μL at 30°C with a Single Tube Protein System 2 (Novagen) according to the manufacturer’s instructions. After synthesis, a 10-μL aliquot of the translation mixture was denatured at 95°C for 5 min in Laemmli buffer (3). The solution was loaded onto a 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and run for 1 h at room temperature in a Mini-PROTEAN II Electrophoresis System (Bio-Rad, Hercules, CA, USA). The gel was dried under vacuum and the radioactivity of the p53 bands counted with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA). A 6-μL aliquot of the same translation mixture was incubated in 20 μL for 1 h at 22°C in the presence of 25 mM HEPES, pH 7.6, 50 mM KCl, 10% glycerol, 5 mM dithiothreitol, 75 μg/mL poly[dI.dC].poly[dI.dC], 300 ng of the activating antibody PAb421 (Oncogene Research Products, Cambridge, MA, USA) (2) and 1.5 × 10-14 mol of double-stranded 32P oligonucleotide. The sequence of the double-stranded oligonucleotide corresponds to the DNA region recognized by wild-type p53 in the WAF1 promoter (1). The oligonucleotides were phosphorylated with the T4 polynucleotide kinase according to Sambrook et al. (4) with a [32P]ATP:oligonucleotide ratio of 10. After incubation, 18 μL of the reaction solution were loaded onto a 4% nondenaturing polyacrylamide gel, which was run at 4°C for 90 min at 200 V. The gel was dried under vacuum, and the regions containing the bound oligonucleotide were excised from the gel. The radioactivity was counted for 10 min in a Tri-Carb 2000 (Packard