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 tagged proteins to enable more speciﬁc puriﬁcation but their ability to bind DNA also enables group speciﬁc afﬁnity puriﬁcation using heparin as a ligand. Heparin is a highly sufonated glycosaminoglycan with the ability to bind a very wide range of biomolecules including:
The structure of heparin is shown in Figure 3.10. Heparin has two modes of interaction with proteins and, in both cases, the interaction can be weakened by increases in ionic strength.
Figure 3.10.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.
Characteristics of chromatography media for puriﬁcation or removal of DNA-binding proteins are shown in Table 3.11.
1 Short term refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. 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.
Puriﬁcation options for Heparin Sepharose 6 Fast Flow chromatography medium and prepacked columns as well as Capto Heparin are shown in Table 3.12.
1 See Appendix 4 to convert ﬂow velocity (cm/h) to volumetric ﬂow rate (mL/min). Maximum operating ﬂow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.
2 1 m diameter column, 20 cm bed height.
Figures 3.11 to 3.13 show examples of conditions used for the puriﬁcation of different DNA binding proteins.
Figure 3.11.Partial puriﬁcation of recombinant HIV-reverse transcriptase on Hi Trap Heparin HP
Figure 3.12.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 3.13.scCro8 puriﬁcation on HiPrep Heparin FF 16/10.
Heparin Sepharose 6 Fast Flow, Heparin Sepharose High Performance
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 2 M.
If used for puriﬁcation or removal of coagulation factors:
Since the heparin acts as an afﬁnity ligand for coagulation factors, it is advisable to include a minimum concentration of 150 mM NaCl in the binding buffer.
If an increasing salt gradient gives unsatisfactory results, use heparin (1 to 5 mg/mL) as a competing agent in the elution buffer.
Remove ionically bound proteins by washing with 0.5 CV of 2 M NaCl for 10 to 15 min.
Remove precipitated or denatured proteins by washing with 4 CV of 100 mM NaOH for 1 to 2 h; or 2 CV of 6 M guanidine hydrochloride for 30 to 60 min; or 2 CV of 8 M urea for 30 to 60 min. Remove hydrophobically bound proteins by washing with 4 CV of 0.1% to 0.5% Tween 20 for 1 to 2 h.
100 mM NaOH (1 w at 20 °C), 50 mM sodium acetate, pH 4.0, 4 M NaCl, 8 M urea, 6 M guanidine hydrochloride.
Wash chromatography media and columns with 50 mM sodium acetate containing 20% ethanol (use approximately 5 CV for packed media) and store at 4 °C to 8 °C.
A ﬂow rate of 0.5 mL/min is recommended for a 1 mL column.
Substances such as denatured proteins that do not elute during regeneration can be removed by cleaning-in-place (CIP) procedures. A recommended CIP procedure for Capto Heparin is 4 CV of 100 mM NaOH with a contact time of 1 to 2 h.
Store unused chromatography media at 4 ºC to 30 ºC in 20% ethanol and 50 mM of sodium acetate.