We have limited experience in protein:DNA crystallization (N=1), but this is what worked for us a few years ago.
Like others have advised, I also suggest using oligos with different ends. We tested two protein constructs, one with a a TEVP cleavable N-terminal His tag and the other with a C-terminal His tag. We used DNA purchased from IDT (standard desalting prep) without further purification. Pairing two protein constructs with several different oligos meant that oligo cost was the limiting reagent. We mixed the the protein and dsDNA in a ratio of protein:dsDNA=1:3, then ran size exclusion chromatography to isolate the complex. Then we concentrated the complex and set up the standard crystal screens and Natrix. The details can be found in the Methods section and supplement of this paper: http://www.ncbi.nlm.nih.gov/pubmed/18586269 J Mol Biol. 2008 Aug 1;381(1):174-88. Structural basis of the transcriptional regulation of the proline utilization regulon by multifunctional PutA. Zhou Y, Larson JD, Bottoms CA, Arturo EC, Henzl MT, Jenkins JL, Nix JC, Becker DF, Tanner JJ. Good luck, Jack On Jan 3, 2014, at 10:23 PM, Acoot Brett wrote: Dear All, For the question, I think for a protein-DNA interaction, the protein may interact with any sequences of DNA, which will give a lot of combination of protein-DNA sequence for crystallization screening. Or do anyone regard to just try the crystallization of the protein-one specific sequence DNA fragment for the trial (for example the DNA sequence with the highest binding affinity)? In another word, does the easiness of the crystallization has relation with how strong the protein interacts with the DNA sequence? Acoot On Saturday, 4 January 2014 11:02 AM, rajakumara eerappa <reera...@gmail.com<mailto:reera...@gmail.com>> wrote: My suggestions are 1 try complementary and non-complementary overhangs which can form Watson-crick and/ or Hoogstein base pairing. 2. If it binds self-complementary duplexes then try them also. 3. Peg conditions with slight acidic pH are more suitable. 4. Divalent cation salts (Ca, Mg, Mn) in crystallization. Wish you good luck Raj On Friday, January 3, 2014, venkatareddy dadireddy wrote: Hi, I'm working on DNA binding protein, looking to co-crystallize protein- DNA complex and have no previous experience. Your suggestion would be very precious on the following queries. 1. My protein is 646 amino acid long and it exists as homodimer. It is also having around 20 amino acid extra sequence from vector. Will vector sequence affect crystallization? 2. Its homologous protein shows good affinity for 31-mer. Shall I use same length of DNA for co- crystallization. 3. What is the length of DNA to be used? 4. What is purity of oligos to be used? Is it HPLC pure or normal desalted ones. I have read on CCP4 mails for screening purpose normal oligos are fine. Please comment on that. 3. Any other suggestions on Protein DNA co- crystallization. Thanks venkat
