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Preparation and Extraction of Insoluble (Inclusion‐Body) Proteins from Escherichia coli

Ira Palmer¹, Paul T. Wingfield¹

¹National Institutes of Health, Bethesda, Maryland

Publication Name:

Current Protocols in Protein Science

Unit Number:

Unit 6.3

DOI:

10.1002/0471140864.ps0603s38

Online Posting Date:

November, 2004

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Paul Wingfield

Abstract

High-level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; alternatively, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates and this unit describes preparation of washed pellets and solubilization of the protein using guanidine×HCl. The extracted protein, which is unfolded, is either directly folded as described in unit 6.5 or further purified by gel filtration in the presence of guanidine×HCl as idescribed here. A support protocol describes the removal of guanidine×HCl from column fractions so they can be monitored by SDS-PAGE.

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Unit Introduction
Basic Protocol 1: Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia Coli
Basic Protocol 2: Medium-Pressure Gel-Filtration Chromatography in the Presence of Guanidine Hydrochloride
Support Protocol: Preparation of Samples Containing Guanidine Hydrochloride for SDS-PAGE
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia Coli

Materials

E. coli cells from fermentation (unit 5.3) containing the protein of interest
Lysis buffer (see recipe)
Wash buffer (see recipe), with and without urea and Triton X-100
Extraction buffer (see recipe)

250- and 500-ml stainless steel beakers
Waring blender
Polytron tissue-grinder homogenizer (Brinkmann)
French pressure cell (e.g., Thermo Electron Corp.; http://www.thermo.com)
Probe sonicator
Beckman J2-21M centrifuge with JA-14 rotor (or equivalent)
Beckman Optima XL-90 ultracentrifuge with 45 Ti rotor (or equivalent)
0.22-µm syringe filters (e.g., Millex from Millipore)
20-ml disposable syringe

Additional equipment for breaking cells, homogenizing cells and pellets and centrifuging at low and high speeds (unit 6.2)

Basic Protocol 2: Medium-Pressure Gel-Filtration Chromatography in the Presence of Guanidine Hydrochloride

Materials

Gel-filtration medium: Superdex 200 PG (preparative grade; Amersham Biosciences)
5% (v/v) ethanol
Gel-filtration buffer (see recipe)
Guanidine×HCl extract of E. coli cells containing the protein of interest (see Basic Protocol 1)

4- to 6-liter plastic beaker
Chromatography column: Amersham Biosciences XK 16/100, 26/100, or 50/100
Packing reservoir: Amersham Biosciences RK 16/26 (for 16- and 26-mm-i.d. columns) and RK 50 (for 50-mm-i.d. column)
Chromatography pump: Amersham Biosciences P-6000 or P-500
Injection valve (to select between sample loop and pump)
UV monitor and fraction collector
Sample loop (volume determined by size of column; also see annotation to step )

NOTE: The various components of the chromatography system (pumps, valves, monitors, and sample loops) listed separately above are supplied as components of the ÄKTAexplorer chromatography system (Amersham Biosciences), which is used to run the XK 50/100 column. The smaller XK columns (2.6 and 2.5 cm i.d.) are run using the ÄKTA-FPLC chromatography system (also from Amersham Biosciences), which is designed for small- to medium-scale work. For further details on this equipment see the manufacturer's literature (e.g., Process Products, Amersham Biosciences).

NOTE: Perform steps to at room temperature. After the column is packed, equilibrate and elute at 4°C.

Support Protocol: Preparation of Samples Containing Guanidine Hydrochloride for SDS-PAGE

Materials

Sample containing the protein of interest
100% and 90% ethanol, 0° to 4°C
1× SDS sample buffer (unit 10.1)

Gilson Pipetman (Rainin Instrument)

Additional reagents and equipment for gel electrophoresis (unit 10.1)

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Figures

Figure 6.3.1

Analysis by SDS-PAGE of fractions from low-speed centrifugation of E. coli cell lysates containing aggregated bovine growth hormone. A 12.5% acrylamide gel of dimensions 12 cm × 16 cm × 1.5 mm was used with the Laemmli buffer system (unit 10.1). Lanes a and g contain molecular weight standards (low-range standards, Bio-Rad) in order of increasing migration distance: phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), hen egg white ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and hen egg white lysozyme (14.4 kDa). After low-speed centrifugation of the clarified lysate and of the washed pellet homogenate (see Basic Protocol 1, steps and ), the supernatants will be cloudy (lane f) and the pellets usually consist of two layers (see Fig. 6.1.5). The bottom layer is inclusion body protein plus unbroken cells (lanes b and c) and the top layer consists of outer membrane and peptidoglycan fragments (lanes d and e). The outer membrane proteins OmpA (35 kDa) and OmpF/C (38 kDa) are indicated by ; and o, respectively. After the washing steps, the growth hormone (marked , 21 kDa) is the major constituent (lanes h and i) together, in this example, with another plasmid-encoded protein, namely kanamycin phosphotransferase (marked , 30.8 kDa), the product of the gene conferring resistance to the antibiotic kanamycin.

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Figure 6.3.2

Gel filtration of an extract containing HIV-1 protease, using Superdex 200 in 4 M guanidine×HCl. Column dimensions, 6 × 60 cm; buffer, 50 mM Tris×Cl (pH 7.5)/4 mM guanidine×Cl/2 mM DTT; flow rate, 5 ml/min (300 ml/hr).The sample has a mass of 10 kDa. Protein fractions 66 to 72 (pool P) was further purified under the same conditions using a Superdex 75 matrix. The inset shows SDS-PAGE analysis of selected fractions. The protein standard markers (lane S) correspond to mass values of 66.2, 45, 30, 21.5, and 14.4 kDa, respectively (migration order top to bottom).

View Image
Figure 6.3.3

Superdex 200 chromatography in guanidine×HCl of SIV gp41^27-149. SDS-PAGE of the numbered fractions is shown in the first inset (upper left); lane “a” contains molecular weight standards (bottom to top: 6.5, 14.4, 21.5, 31, 45, 66.2 kDa), and the purified protein migrates close to the 14.4 kDa standard. Lane “b” represents starting material loaded to column corresponding to guanidine×HCl-extracted inclusion bodies. Protein in the main peak (fractions 5 to 7) marked with arrow was used for protein folding after removal of guanidine×HCl by reversed-phase chromatography. The second inset (upper middle) refers to protein expression in minimal medium; lane A contains the same molecular weight standards as lane “a” in the first inset; lanes B and C correspond to insoluble protein and purified protein, respectively. Protein is labeled with ¹⁵N and ¹³C for NMR analysis. A summary of the protein purification is indicated in the right-hand part of the figure. Adapted from Wingfield et al. (1997).

View Image

Literature Cited

Literature Cited
	Anfinson, C.B. 1973. Principles that govern the folding of protein chains. Science 181:223-230.
	Belew, M., Fohlman, J., and Janson, J.-C. 1978. Gel filtration on Sephacryl S-200 superfine in 6 M guanidine×HCl. FEBS Lett. 91:302-304.
	Creighton, T.E. 1993. Proteins: Structures and Molecular Properties, 2nd ed., pp. 293-296. W.H. Freeman, New York.
	Cowley, D.J. and Mackin, R.B. 1997. Expression, purification and characterization of recombinant human proinsulin. FEBS Letters 402:124-130.
	Darby, N.J. and Creighton, T.E. 1990. Folding proteins. Nature 344:715-716.
	Daniel, R.M., Dines, M. and Petach. 1996. The denaturation and degradation of stable enzymes at high temperature. Biochem J. 317:1-11.
	De Bernardez Clark, E., Schwartz, E. and Rudolph, R. 1999. Inhibition of aggregation side reactions during in vitro protein folding. Methods in Enzymology 309:217-236.
	Falson, P. 1992. An efficient procedure to dialyze volumes in the range of 10-200 ìl. BioTechniques 13:20.
	Fersht, A. 1999. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman, New York.
	Fink, A.L. 1998. Protein aggregation: Folding aggregates, inclusion bodies and amyloid. Folding and Design 3:R9-R23.
	Fish, W.W., Mann, K.G., and Tanford, C. 1969. The estimation of polypeptide chain molecular weights by gel filtration in 6 M guanidine hydrochloride. J. Biol. Chem. 244:4989-4994.
	Frangioni, J.V. and Neel, B.G. 1993. Solubilzation and purification of enzymatically glutathione S-transferase (pGEX) fusion proteins Anal. Biochem. 210:179-187.
	Ghelis, C. and Yon, Y. 1982. Simulation of protein folding: Studies of in-vitro denaturation-renaturation. In Protein Folding (B. Horecker, ed.) pp. 225-243. Academic Press, San Diego.
	Iyer, K.S. and Klee, W. 1973. Direct spectroscopic measurement of the rate of reduction of disulfide bonds J. Biol. Chem. 248:707-710.
	Kane, J.K. and Hartley, D.L. 1991. Properties of recombinant protein-containing inclusion bodies in E. coli. In Purification and Analysis of Recombinant Proteins (R. Seetharam and S.K. Sharma, eds.) pp. 121-145. Marcel Dekker, New York.
	Langley, K.E., Berg, T.F., Strickland, T.W., Fenton, D.M., Boone, T.C., and Wypych, J. 1987. Recombinant-DNA-derived growth hormone from Escherichia coli: Demonstration that the hormone is expressed in the reduced form, and isolation of the hormone in the oxidized, native form. Eur. J. Biochem. 163:313-321.
	Maachupalli-Reddy, J., Kelley, B.D. and De Bernardez Clark, E. 1997. Effect of inclusion body contaminants on the oxidative renaturation of hen white lysozyme. Biotech. Prog. 13:144-150
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	Oberg, K., Chrunyk, B.A., Wetzel, R., and Fink, A.L. 1994. Nativelike secondary structure in interleukin-1 inclusion bodies by attenuated total reflectance FTIR. Biochemistry 33:2628-2634.
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Author Notes

Submitted by: Paul Wingfield

On: August 18, 2009

Recombinant proteins accumulating in the insoluble phase (low speed centrifugation pellets) may be classical inclusion body type protein or may be non-specifically associated with the pelleting material. The distribution of soluble and insoluble protein in the bacterial lysates can be influenced, for example, by induction temperatures and rate of expression etc. Sometimes it may be the best thing to take a small amount of soluble protein and run with it rather than to attempt to work with the larger amount of insoluble protein which will require denaturation to extract it and folding at some stage to activate it. It has been shown that a fraction of inclusion body protein may be natively folded and growth temperature may influence this proportion (Biotech. Bioeng (2007) 96: 1101; see also for comments Nature Biotech (2007) 25:718). As interesting as this may be, the fact remains that protein which is associated with the insoluble fraction of cell lysates has to be extracted with some level of persuasion: in the this unit we use guanidine-HCl to ensure both a high extraction efficiency and, usually, that the protein is physically homogenous (monomeric). We use gel filtration to fractionate the extracted protein and this usually works well and often we follow this by directly applying pooled protein to a reverse phase column (source RP15). The RP-eluted protein can be introduced into several folding scenarios. If the protein is Hist-tagged the protein extract can be directly applied to a Ni-affinity column although prior gel filtration clean-up may give better results.

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vbc

Posted on February 3, 2010 - 12:57pm

Once you've clarified the lysed E.coli cell suspension and are ready to wash the pellets, keep in mind that complete homogenization of the pellet is important to wash out soluble proteins and cellular components. Removal of cell wall and outer membrane material can be improved by increasing the amount of wash solution to 10 ml per gram cells.

The concentration of urea and Triton X-100 in the wash buffer can be varied. The urea concentration is usually between 1 and 4 M; higher concentrations may result in partial solubilization of the recombinant proteins. The usual detergent concentration is 0.5% to 5%. Triton X-100 will not solubilize inclusion body proteins; it is included to help extract lipid and membrane-associated proteins.

Dhaval (not verified)

Posted on February 3, 2010 - 2:39am

can u give me 4-5 strategy for Inclusion bodies washing?