Performing a Separation of DNA Binding Proteins with Cytiva Products Based on Heparin

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:

• DNA binding proteins such as initiation factors, elongation factors, restriction endonucleases, DNA ligase, DNA and RNA polymerases.
• Serine protease inhibitors such as antithrombin III, protease nexins.
• Enzymes such as mast cell proteases, lipoprotein lipase, coagulation enzymes, superoxide dismutase.
• Growth factors such as fibroblast growth factor, Schwann cell growth factor, endothelial cell growth factor.
• Extracellular matrix proteins such as fibronectin, vitronectin, laminin, thrombospondin, collagens.
• Hormone receptors such as oestrogen and androgen receptors.
• Lipoproteins.

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.

1. In its interaction with DNA binding proteins heparin mimics the polyanionic structure of the nucleic acid.
2. In its interaction with coagulation factors such as antithrombin III, heparin acts as an affinity ligand.

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

Purification Options

	Binding capacity	Maximum operating flow	Comments
HiTrap Heparin HP	Bovine antithrombin III, 3 mg/column Bovine antithrombin III, 15 mg/column	4 mL/min (1 mL column)	Prepacked columns.
		20 mL/min (5 mL column)
HiPrep 16/10 Heparin FF	Bovine antithrombin III, 40 mg/column	10 mL/min	Prepacked 20 mL column.
Heparin Sepharose 6 Fast Flow	Bovine antithrombin III, 2 mg/mL medium	400 cm/h*	Supplied as a suspension ready for column packing.
* See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.

Purification Examples

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.

Performing a Separation

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

1. Equilibrate the column with 10 column volumes of binding buffer.
2. Apply the sample.
3. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A_{280 nm}).
4. Elute with 5–10 column volumes of elution buffer using a continuous or step gradient from 0–100% elution buffer.

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.

Cleaning

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.

Media Characteristics

	Ligand density	Composition	pH stability*	Mean particle size
HiTrap Heparin HP	10 mg/mL	Heparin coupled to  Sepharose High Performance  by reductive amination to give a stable attachment even in  alkaline conditions.	Short term 5–10 Long term 5–10	34 µm
Heparin Sepharose 6 Fast Flow   HiPrep 16/10 Heparin FF	5 mg/mL	Heparin coupled to Sepharose 6 Fast Flow by reductive amination to give a stable attachment even in alkaline conditions.	Short term 4–13 Long term 4–12	90 µm
* Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.

Chemical Stability

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.

Storage

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.