Chromatin Protocols [Methods In Molec Bio, Vol 119] - P. Becker (Humana) WW.pdf - książki - Bio Med 06 - bibiariel

Methods in Molecular Biology T TM

VOLUME 119

Chromatin

Edited by

Peter B. Becker

HUMANA PRESS

Methods in Molecular Biology

Chromatin

Protocols

Edited by

Peter B. Becker

Nucleosome Reconstitution

Expression and Purification of Recombinant

Histones and Nucleosome Reconstitution

Karolin Luger, Thomas J. Rechsteiner, and Timothy J. Richmond

1. Introduction

In vitro studies on nucleosome core particles (NCPs) and nucleosomes have

generally been limited to the use of histone proteins isolated from chromatin.

Numerous reliable and well-established methods have been described of

obtaining single histone proteins in significant quantity (e.g., refs. 1 and 2 , and

references therein). Briefly, the histone complexes (histone octamer, or his-

tone tetramer and histone dimer) are isolated from “long chromatin,” which is

extracted from nuclei. The histone complexes can be further fractionated into

individual histone proteins. This approach suffers from several disadvantages.

First, the procedure is time-consuming and depends on the availability of fresh

tissue or blood from the organism of choice. Second, histone proteins isolated

from natural sources are often degraded by contaminating proteases (3) . Third,

histone isotypes and posttranslational modifications of histone proteins give

rise to heterogeneity. The extent of heterogeneity and modification strongly

depend on the type and developmental state of the tissue from which chromatin

is isolated and can vary significantly between different batches. Fourth, and

most important, only naturally occurring histone proteins can be obtained by

this method.

The availability of large amounts of naturally occurring mutants, or of new

site-directed mutants of the highly conserved histone proteins, will be

extremely valuable in our attempts to reconcile the observed functions and

biophysical properties of the NCP with the recently determined atomic struc-

ture (4) . The ability to express all four histone proteins in bacteria has allowed

us to develop a method for the mapping of nucleosome position to base pair

From: Methods in Molecular Biology, Vol. 119: Chromatin Protocols

Edited by: P. B. Becker © Humana Press Inc., Totowa, NJ

Luger, Rechsteiner, and Richmond

resolution (5) and has been instrumental in the structure determination of the

NCP at high resolution (4) . In comparison to yeast expression systems, yields

are high, protease activity is low, and purification does not rely on the presence

of histidine-tags or other fusions (6 , 7) .

This section describes the overexpression of histones H2A, H2B, H3, and

H4, both as full length proteins and corresponding trypsin-resistant “globular

domains” (as defined in [8] ). A simple and efficient purification protocol yields

large amounts of homogenous protein in denatured form. The methods for

refolding and purification of histone octamer and for assembly and purifica-

tion of nucleosome core particles using 146 bp of DNA are described, together

with a protocol for a high-resolution gel shift assay to monitor the purity and

homogeneity of the final core particle preparation.

2. Materials

2.1. Histone Expression

1. pET-histone expression plasmids (2) and transformation-competent cells of the

expression strain BL21(DE3) pLysS (9) .

2. 2X TY-AC media: 16% (w/v) bacto-tryptone, 10% (w/v) yeast extract, and 5% (w/v)

NaCl, supplemented with 100

g/L ampicillin and 25

g/L

chloramphenicol.

4. IPTG: 0.4 M Isopropyl-ß- D -thiogalactopyranoside in water; pass through 0.2-

sterile filter, store frozen in aliquots.

5. Wash buffer: 50 m M Tris-HCl, pH 7.5, 100 m M NaCl, 1 m M Na-EDTA, 1 m M

benzamidine. Shortly before use, add 5 m M 2-mercaptoethanol.

2.2. Histone Purification

1. Wash buffer: as in Subheading 2.1. , item 5 .

2. TW buffer: wash buffer with 1% (v/v) Triton X-100.

3. Unfolding buffer: 7 M guanidinium HCl, 20 m M Tris-HCl, pH 7.5, 10 m M DTT.

Pass through 0.4-

m filters before use.

6. SAU-200: 7 M urea (deionized), 20 m M sodium acetate, pH 5.2, 0.2 M NaCl, 5 m M

2-mercaptoethanol, 1 m M Na-EDTA. Pass through 0.4-

m filters before use.

7. SAU-600: 7 M urea (deionized), 20 m M sodium acetate, pH 5.2, 0.6 M NaCl, 5 m M

2-mercaptoethanol, 1 mM Na-EDTA. Pass through 0.4-

m filters before use.

8. Gel filtration column XK-50 (Pharmacia, Uppsala, Sweden), packed with

Sephacryl S-200 high-resolution gel filtration resin (Pharmacia). Gel bed: 5-cm

diameter, 75-cm height.

g/L chloramphenicol.

3. AC agar plates: 10% (w/v) bacto-tryptone, 5% (w/v) yeast extract, 8% (w/v) NaCl,

and 1.5 % (w/v) Agar, supplemented with 100

m filters before use.

4. Amberlite MB3 or similar ion exchange resin for batch deionization of urea stock

solutions.

5. SAU-1000: 7 M urea (deionized), 20 m M sodium acetate, pH 5.2, 1 M NaCl, 5 m M

2-mercaptoethanol, 1 m M Na-EDTA. Pass through 0.4-

Nucleosome Reconstitution

9. An HPLC system equipped with a TSK SP-5PW HPLC column, 2.15 cm

15.0 cm

(Toyo Soda Manufacturing Company, Tokyo, Japan).

10. Dialysis tubing, molecular weight cutoff 6–8 kDa, widths 5 cm and 2.5 cm. Prepare

according to the supplier and rinse thoroughly with distilled water before use.

11. Standard SDS-PAGE equipment; 18% SDS gels for analysis of protein fractions (10) .

2.3. Histone Octamer Reconstitution

1. Unfolding buffer: as in Subheading 2.2.3.

2. Refolding buffer: 2 M NaCl, 10 m M Tris-HCl, pH 7.5, 1 m M Na-EDTA, 5 m M

2-mercaptoethanol.

3. Gel filtration column HiLoad 16/60 Superdex 200 prep grade (Pharmacia),

equipped with UV-detector and fraction collector; at 4

2.4. Nucleosome Core Particle Reconstitution

1. Purified histone octamer at a concentration of at least 0.75 mg/mL, in refolding buffer.

2. DNA of length greater than 138 bp, with a known concentration (at least 3 mg/mL).

3. A peristaltic pump with a double pump head, capable of maintaining a flow rate

of approx 2–6 mL/min (e.g., Gilson Minipuls 3 peristaltic pump, equipped with

tubing with 2.5 mm inner diameter; Gilson Medical Electronics SA, Villier-leBel,

France); or two peristaltic pumps.

4. A reconstitution flask with connected tubing, as shown in Fig. 1 .

5. Buffers for reconstitution:

RB-high: 2 M KCl, 10 m M Tris-HCl, pH 7.5, 1 m M EDTA, 1 m M DTT

RB-low: 0.25 M KCl, 10 m M Tris-HCl, pH 7.5, 1 m M EDTA, 1 m M DTT.

2.5. Nucleosome Core Particle Purification

2.5.1. Purification by HPLC-Ion Exchange Chromatography

1. TES-250: 0.25 M KCl, 10 m M Tris-HCl, pH 7.5, 0.5 m M EDTA.

2. TES-600: 0.6 M KCl, 10 m M Tris-HCl, pH 7.5, 0.5 m M EDTA.

3. A HPLC apparatus equipped with a TSK DEAE-5PW HPLC column, 2.15

15.0 cm,

or a TSK DEAE-5PW HPLC column, 7.5

75 mm (Toyo Soda Manufacturing);

preferrably at 4

2.5.2. Purification by Preparative Gel Electrophoresis;

High-Resolution Gel Shift Assay

1. Model 491 Prep Cell (Bio-Rad Laboratories, Richmond, CA) with a standard

power supply, connected to a UV detector and a fraction collector, and equipped

with a peristaltic pump.

2. Gel running buffer: 0.20X TBE (1X TBE: 89 m M Tris-HCl, 89 m M boric acid,

and 2.5 m M EDTA).

C ( see Note 7 ).

4. Standard SDS-PAGE equipment ( see Subheading 2.2. , step 11 ).

5. Concentration device suitable for 1–25 mL volumes (e.g., Sartorius ultrathimble,

Sartorius AG, Göttingen, Germany; or Centricon 10, Amicon AG, Beverley, MA).

Luger, Rechsteiner, and Richmond

Fig. 1. Schematic drawing of the experimental apparatus for reconstitution. We use

a 500-mL glass flask as a reconstitution vessel, and a peristaltic pump with a four-

channel head. Standard glass tubes are bent in the appropriate manner and are con-

nected by silicone tubing. The reconstitution vessel contains RB-high to start.

4. Dialysis membrane (molecular weight cutoff: 6–8 kDa), cut to a circle with a

radius of 3 cm. Prepare according to the supplier and rinse thoroughly with dis-

tilled water before use.

5. Concentration device as specified in Subheading 2.3.5.

6. Storage buffers: TCS buffer:

20 m M Tris-HCl, pH 7.5, 1 m M EDTA, 1 m M DTT

CCS buffer: 20 m M K-Cacodylate, pH 6.0, 1 m M EDTA.

3. Methods

3.1. Histone Expression

Expression plasmids for the individual histone proteins and their N-termi-

nally truncated versions, based on the T7-expression system (9) , have been

described previously (2) . High level expression of the Xenopus laevis histone

genes for H2A, H2B, and H3 does not necessitate adaptation of the codon

usage, despite the presence of several codons with low usage in Escherichia

coli . These proteins can be expressed with similar efficiencies as N-terminal

3. Acrylamide stock solution: 29.5% acrylamide, 0.5% bis- acrylamide in water.

Deionized by stirring with Amberlite MB3, and stored at 4

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