DNA binding proteins form an extremely diverse class of proteins sharing a single characteristic, their ability to bind to DNA. Functionally the group can be divided into those responsible for the replication and orientation of the DNA such as histones, nucleosomes and replicases and those involved in transcription such as RNA/DNA polymerases, transcriptional activators and repressors and restriction enzymes. They can be produced as fusion proteins to enable more speciﬁc puriﬁcation (page 42, GST fusion proteins), but their ability to bind DNA also enables group speciﬁc afﬁnity puriﬁcation using heparin as a ligand. Heparin is a highly sulphated glycosaminoglycan with the ability to bind a very wide range of biomolecules including:
The structure of heparin is shown in Figure 1. Heparin has two modes of interaction with proteins and, in both cases, the interaction can be weakened by increases in ionic strength.
Figure 1. Structure of a heparin polysaccharide consisting of alternating hexuronic acid (A) and D-glucosamine residues (B). The hexuronic acid can either be D-glucuronic acid (top) or its C-5 epimer, L-iduronic acid (bottom).
R1 = -H or -SO3–, R2 = -SO3– or -COCH3
Figures 2, 3 and 4 show examples of conditions used for the puriﬁcation of different DNA binding proteins.
Figure 2. Partial purifcation of recombinant HIV-reverse transcriptase on HiTrap Heparin HP.
Figure 3. Partial puriﬁcation of the recombinant DNA binding Oct-1 protein (courtesy of Dr Gunnar Westin, University Hospital, Uppsala, Sweden) using HiTrap Heparin HP, 5 mL.
Figure 4. scCro8 puriﬁcation on HiPrep 16/10 Heparin FF.
Binding buffers: 20 mM Tris-HCl, pH 8.0 or 10 mM sodium phosphate, pH 7.0
Elution buffer: 20 mM Tris-HCl, 1–2 M NaCl, pH 8.0 or 10 mM sodium phosphate, 1–2 M NaCl, pH 7.0
Modify the selectivity of heparin by altering pH or ionic strength of the buffers. Elute using a continuous or step gradient with NaCl, KCl or (NH4)2SO4 up to 1.5–2 M.
Remove ionically bound proteins by washing with 0.5 column volume 2 M NaCl for 10–15 minutes.
Remove precipitated or denatured proteins by washing with 4 column volumes 0.1 M NaOH for 1–2 hours or 2 column volumes 6 M guanidine hydrochloride for 30–60 minutes or 2 column volumes 8 M urea for 30–60 minutes.
Remove hydrophobically bound proteins by washing with 4 column volumes 0.1% – 0.5% Triton X-100 for 1–2 hours.
0.1 M NaOH (1 week at +20 °C), 0.05 M sodium acetate, pH 4.0, 4 M NaCl, 8 M urea, 6 M guanidine hydrochloride.
Wash media and columns with 0.05 M sodium acetate containing 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.