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Folding and Purification of Insoluble (Inclusion Body) Proteins from Escherichia coli

Paul T. Wingfield¹, Ira Palmer¹, Shu‐Mei Liang²

¹National Institutes of Health, Bethesda, Maryland
²North American Vaccine Corp., Beltsville, Maryland

Publication Name:

Current Protocols in Protein Science

Unit Number:

Unit 6.5

DOI:

10.1002/0471140864.ps0605s00

Online Posting Date:

May, 2001

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Paul Wingfield

Abstract

Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed and the are aggregates, (2) the cell wall and outer membrane components of the aggregates are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidineHCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an “oxido-shuffling” buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein which is then partially purified by gel filtration before folding, and then the protein is folded and oxidized by simple dialyzed against water. A Support Protocol is included for rapidly determining the amount of folded protein that contains the correct disulfide linkage pattern. Finally, folding and purification of a fusion protein is described using metal-chelate affinity chromatography.

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Unit Introduction
Basic Protocol 1: Folding and Purification of Bovine Growth Hormone
Basic Protocol 2: Folding and Purification of Human Interleukin 2
Support Protocol: Resolution of Native and Misfolded Forms of hIL-2 by RP-HPLC
Basic Protocol 3: Folding and Purification of a Histidine-Tagged Protein: HIV-1 Integrase
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Folding and Purification of Bovine Growth Hormone

Materials

E. coli cells expressing BGH: ~50 g wet weight from a 1.5-liter fermentation (unit 5.3), and stored as a flattened paste in a sealed polyethylene bag at –80°C
BGH break buffer A (see recipe), 4°C
BGH break buffer B (see recipe), 4°C
BGH wash buffer A (see recipe), 4°C
BGH wash buffer B (see recipe), 4°C
BGH extraction buffer (see recipe), 4°C
BGH folding buffer A (see recipe), 4°C
BGH folding buffer B (see recipe), 4°C
2 M HCl
BGH column buffer A (see recipe), 4°C
DEAE Sepharose CL-4B ion-exchange resin (Pharmacia Biotech)
Sephadex G-100 gel filtration resin (Pharmacia Biotech)
BGH column buffer B (see recipe), 4°C

Blender (e.g., Waring; ³1 liter capacity)
Tissue homogenizer (e.g., Polytron, Brinkmann)
Beckman J2-21M centrifuge and JA-20 rotor (or equivalent)
Sonicator, ³400 W, with sound enclosure (Branson or equivalent)
0.7-µm glass-microfiber filters, 4.7-cm diameter (Whatman GF/F) with vacuum filtration apparatus (optional)
Spectra/Por 1 dialysis tubing, 40-mm diameter (MWCO 6000 to 8000; Spectrum)
5 × 50–cm and 5 × 100–cm glass chromatography columns with adjustable flow adaptors (Pharmacia Biotech)
Stirred cell with Diaflo PM 10 ultrafiltration membrane (Amicon)
Millex-GV 0.22-µm filter units (Millipore)

Additional reagents and equipment for cell breakage using a French press (unit 6.2), dialysis (appendix 3B), ion-exchange chromatography (unit 8.2), gel-filtration chromatography (unit 8.3), and SDS-PAGE (unit 10.1)

NOTE: All steps are carried at 4°C unless otherwise stated.

Basic Protocol 2: Folding and Purification of Human Interleukin 2

Materials

hIL-2 break buffer (see recipe), 4°C
E. coli cells expressing hIL-2: ~20 g wet weight from a 3-liter fermentation (unit 5.3), and stored as a flattened paste in a sealed polyethylene bag at –80°C
Sucrose
Lysozyme (Worthington)
hIL-2 wash buffer: 0.75 M guanidine×HCl/1% (w/v) Tween 20 (prepare immediately before use), 4°C
PBS (appendix 2E), 4°C
10% and 20% (v/v) acetic acid, 4°C (prepare fresh from glacial acetic acid)
Sephadex G-100 gel-filtration resin (Pharmacia Biotech)
Acetonitrile (HPLC grade)
Trifluoroacetic acid (TFA; HPLC grade)
7 × 250–mm 300-Å octyl Aquapore RP-300 semiprep column (Brownlee column; Thomson Instrument)
RP-HPLC solvent A (see recipe), room temperature
RP-HPLC solvent B (see recipe), room temperature
25 mM acetic acid, 4°C

Tissue homogenizer (e.g., Polytron, Brinkmann)
30°C water bath
Sorvall RC-5C centrifuge with SS-34 rotor (or equivalent)
2.6 × 100–cm glass chromatography column
Spectra/Por 3 dialysis tubing, 11.5- and 45-mm diameters (MWCO 3500; Spectrum)
Sterivex-GS 0.22-µm filter units (Millipore)
HPLC system with pumps, UV detector, and fraction collector (Waters)

Additional reagents and equipment for cell breakage using a French press (unit 6.2), gel-filtration chromatography (unit 8.3), and dialysis (appendix 3B)

Support Protocol: Resolution of Native and Misfolded Forms of hIL-2 by RP-HPLC

Additional Materials (also see Basic Protocol 2)

0.46 × 10–cm SynChropak RP-P C18 column (Thomson Instrument)
RP-HPLC solvent C (see recipe)
25 mM acetic acid
Purified folded hIL-2 solution (see Basic Protocol 2)

Basic Protocol 3: Folding and Purification of a Histidine-Tagged Protein: HIV-1 Integrase

Materials

E. coli cells expressing HIV-1 integrase: 100 g wet weight from a 3.5-liter fermentation (unit 5.3), stored as a flattened paste in a sealed polyethylene bag at –80°C
IN break buffer (see recipe), 4°C
Suspension buffer: IN break buffer (see recipe) prepared without lysozyme, 4°C
IN extraction buffer (see recipe), 4°C
6 × 60–cm Superdex 200 prep grade prepacked gel-filtration column (Pharmacia Biotech)
IN column buffer A (see recipe), 4°C
Ni-NTA-Sepharose CL-6B MCAC resin (Qiagen)
IN column buffer B (see recipe), 4°C
IN column buffer C (see recipe), 4°C
IN column buffer D (see recipe), 4°C
6 × 60–cm Superdex 75 prep grade prepacked gel-filtration column (Pharmacia Biotech)
IN column buffer E (see recipe), 4°C
4 M guanidine×HCl/5 mM DTT (4°C)
IN folding buffer (see recipe), 4°C
50 mM Tris×Cl (pH 7.5 at 4°C)/10 mM CHAPS, 4°C
2000 NIH unit/ml thrombin (see recipe)
IN column buffer F (see recipe), 4°C
9 M urea, 4°C
IN column buffer G (see recipe), 4°C
p-aminobenzamidine immobilized on Sepharose 6B (Pierce, Pharmacia Biotech, or Sigma)
0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; Boehringer Mannheim or ICN Biomedicals)

Blender (e.g., Waring; ³1 liter capacity)
1-liter steel beaker
Sonicator, ³400 W, with sound enclosure (Branson or equivalent)
Centrifuge with Beckman J-14 rotor (or equivalent)
Tissue homogenizer (e.g., Polytron, Brinkmann)
Ultracentrifuge with Beckman 45Ti rotor (or eqivalent)
5 × 50–cm chromatography column with adjustable flow adaptors
Stirred cells (400-ml and 2-liter capacities) with Diaflo PM 10 ultrafiltration membranes (Amicon)
Peristaltic pump
Millex-GV 0.22-µm pore size filter units (Millipore)
28°C water bath
1 × 10– to 1 × 20–cm chromatography column

Additional reagents and equipment for gel-filtration chromatography (unit 8.3) and SDS-PAGE (unit 10.1)

NOTE: All steps are carried at 4°C unless otherwise stated.

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Figures

Figure 6.5.1

SDS-PAGE of bovine growth hormones on 12.5% polyacrylamide gel. Lane A, BGH-expressing E. coli cells minus the expression vector; lanes B and C, BGH-expressing E. coli cells with A-4 and A-9 BGH expression vectors, respectively; lane D, purified recombinant A-9 BGH; lane E, BGH purified from pituitary (supplied by A.F. Parlow, UCLA); lane F, purified recombinant A-9 BGH with no reductant in sample buffer; lane G, BGH purified from pituitary with no reductant in sample buffer. In lane E (pituitary BGH), the two bands correspond to full-length protein and protein truncated at the N-terminus by 4 residues. It can be seen that the bottom band has the same mobility as E. coli extracts containing the A-4 BGH construct. In lane G, it it may be noted that the two bands are not resolved under nonreducing conditions.

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Figure 6.5.2

Chromatograms illustrating peaks produced by (A) correctly folded hIL-2 (in absence of denaturant); (B) correctly folded hIL-2 (in presence of denaturant but at pH 3.5, which is too low for disulfide-bond exchange); (C) scrambled hIL-2 isomers (resulting from denaturant treatment at pH 8.5); (D) unfolded hIL-2 (resulting from denaturant/reductant treatment).

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Figure 6.5.3

Setup for folding of HIV-1 integrase by dilution into buffer.

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Figure 6.5.4

Results of SDS-PAGE of H1V-1 integrase^50-212 on a 12.5% polyacrylamide gel stained with Coomassie blue. Lane A, recombinant HIV-1 integrase^50-212; lane B, recombinant HIV-1 integrase^50-212 with N-terminal His tag; lane C, extract of HIV-1 integrase–expressing E. coli cells used for purification.

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Literature Cited

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Author Notes

Submitted by: Paul Wingfield

On: August 18, 2009

We will soon be introducing a new Chapter on protein folding with separate units dealing with recombinant protein folding issues. Further, in Chapter 6 we will introduce more units dealing with recovery of active proteins from inclusion bodies and other units dealing with methods which may prevent aggregates forming in the first place, for example, co-expression systems with chaperones.