Partial purification of DNA binding proteins using HiTrap Heparin HP

Transcription

1 application note Affinity chromatography Partial purification of DNA binding proteins using HiTrap Heparin HP Abstract This work describes partial purification of three different DNA binding proteins, i.e. H-NS, RNA polymerase d Oct-1, using pre-packed HiTrap Heparin HP 5 ml columns in the initial chromatographic step. H-NS was purified directly from a bacterial lysate using heparin affinity chromatography d gel filtration as the only chromatographic steps. The protein was shown to be 70% pure by SDS-PAGE. RNA polymerase was purified using gel filtration d ion exchge chromatography after the heparin step. SDS-PAGE alysis showed the polymerase to be 90% pure. Recombint hum Oct-1 was shown to bind to HiTrap Heparin HP, but was not further purified. The results show that HiTrap Heparin HP is a suitable column for the binding of both native d recombint DNA binding proteins directly from bacterial lysates. Introduction DNA binding proteins are of prime interest to my researchers because of their key role in replication, recombination d expression of DNA. My DNA binding proteins are involved in the trscription process occurring in all living cells. DNA binding proteins c be divided into two functional classes: those responsible for the replication d orientation of the DNA, e.g. nucleosomal histones, replicases, d those directly involved in the trscription function, e.g. RNA polymerases, trscriptional activators d repressors. Both classes have in common their capacity to bind DNA with varying degrees of specificity. Some proteins recognise specific DNA sequences whereas others bind to specific structures of DNA (1). My different methods have been proposed for the initial isolation of DNA binding proteins. Stdard purification methods such as ion-exchge chromatography, usually DEAE-cellulose or phosphocellulose, may be used, but are not very selective (2, 3, 12). Recombint species are often expressed with affinity tag at their N- or C-terminus to simplify purification. These tags c however affect the interaction of DNA proteins with their target sequence (4). High-specificity affinity chromatography using immobilised DNA with a sequence recognised by the protein to be purified is often used (5, 6). This technique is however most suitable as a final purification step, as the ligds are often susceptible to degradation by enzymes, e.g. RNases d DNases, occurring in cellular extracts. My DNA binding proteins have been purified using biomimetic affinity ligds, e.g. heparin immobilised to different matrixes (5, 7, 8). Binding to heparin involves both charge d ligd specificity d therefore gives a general group separation of DNA binding proteins. The structure d negative charge of heparin enable it to mimic DNA in its overall binding properties. Also, since heparin is not susceptible to bacterial RNases d DNases it is ideal ligd to use for initial purification of DNA binding proteins from bacterial lysates. In this work we have evaluated the suitability of HiTrap Heparin HP for the initial purification of three different DNA binding proteins. H-NS (histone-like nucleoid structuring protein) is one of the most abundt proteins associated with the Escherichia coli (E. coli) nucleoid. RNA polymerase is a trscribing enzyme responsible for the synthesis of all RNA species in prokaryotes. Oct-1 is octamer-binding trscription factor, which stimulates trscription of a hum histone H2b gene. The proteins H-NS d RNA polymerase are both found naturally in E. coli d have molecular weights of M r d M r respectively (9, 10). Oct-1 is a basic protein with a molecular weight, M r (11) AB, p1

2 Material d methods All chemicals used were of alytical grade. Pefabloc (AEBSF) is a non-toxic protease inhibitor purchased from Boehringer Mnheim. Rabbit ti-oct hum-1 was from Sta Cruz Biotechnology, Inc. Anti-rabbit IgG (γ -chain specific) Alkaline Phosphatase Conjugate d insoluble Alkaline Phosphatase Substrate were from SIGMA. Nitro-cellulose (E) filters were from Schleicher & Schuell. Centricon 10 concentrators were from Amicon. Millex HA filters were from Millipore. HiTrap Heparin HP d HiTrap Q HP columns, Superdex 75 HR 10/30 d Superdex 200 HR 10/30 columns, PD-10, FPLC System, PhastSystem, PhastTrsfer d PhastGel were all from Amersham Biosciences. The E. coli strain BL21 plyss expressing the recombint Oct-1 protein was kindly provided by Dr Gunnar Westin, University Hospital, Uppsala, Sweden. The E. coli strain MM 294 containing H-NS was a kind gift from Dr Sylvie Rimsky d E. coli MRE 600 with RNA polymerase was a kind gift from Dr Malcolm Buckle, both from the Pasteur Institute, Paris, Frce. Chromatography All chromatography was done using FPLC System. 1 mm Pefabloc) prior to sonication. The cells were sonicated on ice d then centrifuged at rpm ( g) at 5 C for 1 hr. The supernatts were saved. RNA polymerase RNA polymerase was precipitated by adding solid ammonium sulphate to the extract to 50% final concentration. The solution was stirred for 30 min on ice. The solution was then centrifuged at rpm ( g) for 90 min at 5 C. The pellet was dissolved in binding buffer (see Fig. 1a). The extract was exchged into binding buffer on a PD-10 column before loading onto HiTrap Heparin HP 5 ml. Oct-1 The extract containing Oct-1 was exchged into binding buffer (see Fig. 2) on a PD-10 column before loading onto HiTrap Heparin HP 5 ml. H-NS Solid ammonium sulphate was added slowly to the extract containing H-NS to 40% final concentration. The solution was stirred for 30 min at room temperature d was then centrifuged at rpm ( g) for 30 min at 5 C. The pellet was dissolved in binding buffer (see Fig. 3a). The extract was exchged into binding buffer on a PD-10 column before loading onto HiTrap Heparin HP 5 ml. Analysis Electrophoresis: All electrophoretic runs were performed on PhastSystem according to the instruction mual. PhastGel Gradient d PhastGel Gradient 8 25 were used. The gels were Coomassie Blue or silver stained. Western Blotting: Electrophoretic trsfer of proteins from a PhastGel to a nitro-cellulose filter was performed using PhastTrsfer d PhastSystem according to the instruction muals. After trsfer, the filter was incubated for 1 hr in room temperature in 10 ml blocking buffer (3% (w/v) nonfat dry milk in PBS, ph 7.4) containing 50 µl of ti-oct-1. The filter was washed five times in wash buffer (PBS, ph 7.4, 0.5% (v/v) Tween 20). The filter was then incubated in room temperature for 1 hr in 20 ml blocking buffer containing 20 µl ti-rabbit IgG. The filter was washed five times in wash buffer before development in 10 ml of alkaline phosphatase substrate (BCIP/NBT) until a purple bd appeared. Sample preparation The different strains of E. coli containing the DNA binding proteins were thawed d suspended in twice the volume of disruption buffer (20 mm Tris-HCl, ph 8, 5% (v/v) glycerol, 1 mm EDTA, 0.25 M NaCl, 20 mm 2-mercaptoethol, AA, p2

3 Results a) Chromatography on HiTrap Heparin HP 5 ml b) Gel filtration on Superdex 200 HR 10/30 Binding buffer: Elution buffer: Sample volume: Gradient: Fraction volume: Extract containing RNA polymerase, filtered through a 0.45 µm Millex HA filter HiTrap Heparin HP 5 ml 10 mm 2-mercaptoethol, 0.1 mm Pefabloc, 0.2 M NaCl 10 mm 2-mercaptoethol, 0.1 mm Pefabloc, 1 M NaCl 35 ml 1 ml/min (30 cm/h) Linear, 0.2 M NaCl to 1 M NaCl in 20 column volumes 3 ml from figure 1a, concentrated on a Centricon 10 concentrator Superdex 200 HR 10/30 Buffer: 10 mm 2-mercaptoethol, 0.1 mm Pefabloc, 0.2 M NaCl Sample volume: 500 µl 0.25 ml/min (19 cm/h) Volume (ml) Volume (ml) using PhastGel 10 15, Coomassie Blue staining Le 1: Low Molecular Weight Calibration Kit (LMW) Le 2: Fraction 17 Le 3: Fraction 15 Le 4: Fraction 13 Le 5: Fraction 11 Le 6: Fraction 9 Le 7: Starting material, E. coli extract, dil. 5x Le 8: LMW Fig. 1b. Purification of RNA polymerase. using PhastGel 10 15, silver staining Le 1: Le 2: Le 3: Le 4: E. coli extract, dil. 60x Starting material, pool from HiTrap Heparin HP LMW Fig. 1a. Purification of RNA polymerase AB, p3

4 c) Ion exchge chromatography on HiTrap Q HP 1 ml Chromatography on HiTrap Heparin HP 5 ml Starting buffer: Elution buffer: Sample volume: Gradient: Fraction volume: from figure 1b HiTrap Q HP 1 ml 10 mm 2-mercaptoethol, 0.1 mm Pefabloc, 0.2 M NaCl 10 mm 2-mercaptoethol, 0.1 mm Pefabloc, 1 M NaCl 1 ml 1 ml/min (158 cm/h) Linear, 0.2 M NaCl to 0.5 M NaCl in 30 column volumes 0.5 ml Binding buffer: Elution buffer: Sample volume: Extract containing Oct-1, filtered through a 0.45 µm Millex HA filter HiTrap Heparin HP 5 ml 10 mm 2-mercaptoethol, 0.1 mm Pefabloc, 0.5 M NaCl 10 mm 2-mercaptoethol, 0.1 mm Pefabloc, 2 M NaCl 30 ml 1 ml/min (30 cm/h) Volume (ml) Volume (ml) M r using PhastGel 10 15, Coomassie Blue staining using PhastGel 10 15, silver staining Le 1: Starting material, pool from Superdex 200, dil. 3x Le 2: Fraction 44 Le 3: Fraction 45 Le 4: LMW Le 5: Fraction 4 Le 6: Fraction 29 Le 1: Le 2: Le 3: Le 4: Starting material, E. coli extract, dil. 4x Flow-through Low Molecular Weight Calibration Kit (LMW) 6 7 Western blotting of the above electrophoresed gel Fig. 1c. Purification of RNA polymerase. Le 1: Le 2: Le 3: Le 4: Starting material Flow-through LMW Fig. 2. Purification of Oct AA, p4

5 Discussion A method is described for partial purification of DNA binding proteins directly from bacterial lysates. RNA polymerase was bound to HiTrap Heparin HP at 0.2 M NaCl d was eluted in a salt gradient (see Fig. 1a). The RNA polymerase pool from HiTrap Heparin HP was further purified by gel filtration on Superdex 200 HR 10/30 (see Fig. 1b). To eliminate the remaining impurities, the pool from the gel filtration step was applied onto a HiTrap Q HP column (see Fig. 1c). Analysis of the eluted fractions confirmed that the protein purified was mainly RNA polymerase core enzyme. Purity was estimated to 90%. Oct-1 d H-NS were bound to HiTrap Heparin HP under conditions using high salt concentrations (see Figs. 2 d 3a), indicating that the proteins bind in a non-ionic but a very specific mner. Oct-1 was eluted from HiTrap Heparin HP in a step gradient. The protein was identified in the eluate using Western blotting as the concentration was too low to be detected by Coomassie Blue staining (Fig. 2). The protein was not further purified. The fractions from HiTrap Heparin HP containing H-NS were pooled (see Fig. 3a) d applied to a Superdex 75 HR 10/30 gel filtration column. The SDS-PAGE in figure 3b shows that the pooled fraction containing H-NS is still contaminated with low molecular weight components. However, H-NS was shown to be 70% pure. The results from this study show that all three DNA binding proteins bind to HiTrap Heparin HP, regardless of molecular mass. HiTrap Heparin HP was shown to be a suitable column to use as a first chromatographic purification step. Conclusions HiTrap Heparin HP is a suitable column to use for the binding of both native d recombint DNA binding proteins directly from bacterial lysates. The accessibility of the matrix to the protein is unaffected by the molecular size of the proteins studied. By using HiTrap Heparin HP as the first chromatographic step combined with a second gel filtration step on a pre-packed Superdex HR 10/30 column, two of the DNA binding proteins studied could be purified to over 70% purity. Acknowledgements We would like to thk Dr Gunnar Westin for providing the Oct-1 clone d Dr Sylvie Rimsky for the E. coli strain containing H-NS. Dr Malcolm Buckle is gratefully acknowledged for the generous gift of the E. coli strain containing RNA polymerase d for his help with alysing the purified protein. We would also like to thk them all for their helpful advice during this work. References 1. DNA-Protein Interactions. Travers, A. (Chapm & Hall, London) Partial purification of a pea seed DNA-binding protein that specifically recognizes 5-methylcytosine. Prep. Biochem. 23 (1993) , Ehrlich, K. C. 3. Trscriptional regulation of the apolipoprotein B100 gene: Purification d characterization of trs-acting factor BRF-2. Mol. Cell. Biol. 12 (1992) , Zhug, H. et al. 4. The histidine tail of recombint DNA binding proteins may influence the quality of interaction with DNA. Anal. Biochem. 234 (1996) , Büning, H. et al. 5. Purification of the sequence-specific trscription factor CTCBF, involved in the control of hum collagen IV genes: subunits with homology to Ku tigen. EMBO J. 14 (1995) , Genersch, E. et al. 6. Micropurification of DNA binding proteins by DNA-affinity chromatography. Technical Note, Amersham Biosciences, code no In vitro trscription close to the melting point of DNA: alysis of Thermotoga maritima RNA polymerase-promoter complexes at 75 C using chemical probes. Nucleic Acids Res. 23 (1995) , Meier, T. et al. 8. EBNA1 c link the enhcer element to the initiator element of the Epstein-Barr virus plasmid origin of DNA replication. J. Virol. 66 (1992) , Middleton, T. d Sugden, B. 9. The chromatin-associated protein H-NS. Biochimie 76 (1994) , Ussery, D. W. et al. 10. Rapid isolation of highly active RNA polymerase from Escherichia coli d its subunits by matrix-bound heparin. Eur. J. Biochem. 60 (1975) 51 55, Sternbach, H. et al. 11. Purification d characterization of OTF-1, a trscription factor regulating cell cycle expression of a hum histone H2b gene. Cell 51 (1987) , Fletcher, C. et al. For more references regarding applications for HiTrap Heparin HP, see: Ordering Information Product Qutity Code No. HiTrap Heparin HP 5 1 ml HiTrap Heparin HP 1 5 ml HiTrap Q HP 5 1 ml HiTrap SP HP 5 1 ml Superdex 75 10/300 GL 1 24 ml Superdex /300 GL 1 24 ml PD-10 Columns HiTrap Desalting 5 5 ml HiPrep 16/10 Heparin FF 1 20 ml Related products Product Qutity Code No. HiTrap Column Guide Affinity Chromatography, Columns d Media Product Profile Affinity Chromatography Hdbook, Principles d methods AB, p5

6 a) Chromatography on HiTrap Heparin HP 5 ml Extract containing H-NS, filtered through a 0.45 µm Millex HA filter HiTrap Heparin HP 5 ml Binding buffer: 10 mm 2-mercaptoethol, 0.1 mm Pefabloc, 0.6 M NaCl Elution buffer: 10 mm 2-mercaptoethol, 0.1 mm Pefabloc, 1 M NaCl Sample volume: 30 ml 1 ml/min (30 cm/h) b) Gel filtration on Superdex 75 HR 10/30 from fig 3a, concentrated on a Centricon 10 concentrator Superdex 75 HR 10/30 Buffer: 10 mm 2-mercaptoethol, 0.1 mm Pefabloc, 0.2 M NaCl Sample volume: 500 µl 0.25 ml/min (19 cm/h) Volume (ml) Volume (ml) using PhastGel 8 25, Coomassie Blue staining using PhastGel 8 25, Coomassie Blue staining Le 1: Le 2: Le 3: Le 4: Starting material, E. coli extract, dil. 10x Flow-through, dil. 5x Low Molecular Weight Calibration Kit (LMW) Le 1: Le 2: Le 3: Le 4: E. coli extract, dil. 10x Starting material, pool from HiTrap Heparin HP LMW Fig. 3b. Purification of H-NS. Fig. 3a. Purification of H-NS. Asia Pacific Tel: Fax: Australasia Tel: Fax: Austria Tel: Fax: Belgium Tel: Fax: Cada Tel: Fax: Central, East, South East Europe Tel: Fax: Denmark Tel: Fax: Finld & Baltics Tel: +358 (0) Fax: +358 (0) Frce Tel: Fax: Germy Tel: Fax: Italy Tel: Fax: Jap Tel: Fax: Latin America Tel: Fax: Middle East d Africa Tel: Fax: Netherlds Tel: Fax: Norway Tel: Fax: Portugal Tel: Fax: Russia & other C.I.S. & N.I.S. Tel: +7 (095) , Fax: +7 (095) South East Asia Tel: Fax: Spain Tel: Fax: Sweden Tel: Fax: Switzerld Tel: Fax: UK Tel: Fax: USA Tel: Fax: HiTrap, HiPrep, Superdex, FPLC, PhastGel d PhastSystem are trademarks of Amersham Biosciences Limited. Amersham d Amersham Biosciences are trademarks of Amersham plc. Pefabloc is a trademark of Pentafam AG. Centricon is a trademark of Amicon Inc. Amicon is a trademark of Millipore Inc. Millex is a trademark of Millipore Corp. Coomassie is a trademark of ICI plc. Tween is a trademark of ICI Americas Inc. Amersham Biosciences AB Björkgat 30, SE Uppsala, Sweden Amersham Biosciences UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, Engld Amersham Biosciences Corp. 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855, USA Amersham Biosciences Europe GmbH Munzinger Strasse 9, D Freiburg, Germy Amersham Biosciences K.K. Sken Building, , Shinjuku-ku, Tokyo , Jap. All goods d services are sold subject to the terms d conditions of sale of the compy within the Amersham Biosciences group that supplies them. A copy of these terms d conditions is available on request. Amersham Biosciences AB 2003 All rights reserved AB, p6