Iozzo Renato V. Proteoglycan Protocols

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214 McQuillan et al.

200 µLB+3.6 mL 2.5% MEM = C (10

–5

)

200 µLC+1.0 mL 2.5% MEM = D (10

–6

)

200 µLD+1.6 mL 2.5% MEM = E (10

–7

)

200 µLE+1.8 mL 2.5% MEM = F (10

–8

)

200 µLF+1.8 mL 2.5% MEM = G (10

–9

)

5. Aspirate media from cells and infect with 0.5 mL of diluted lysate per well (plate dilu-

tions E, F, G in duplicate).

6. Incubate for 2 h at 37°C, rocking every 15 min.

7. Overlay with 1.5 mL of 2.5% DMEM per well. Incubate for 2 d.

7. Day 4: Aspirate medium and add 0.5 mL of crystal violet for each well. Incubate at room

temperature for 5 min. Aspirate and allow wells to air dry.

8. Count plaques. There should be 30–90 plaques per well to give an accurate titration. The

titer of the virus is calculated and expressed as “plaque-forming units” (pfu) per milliliter

of virus stock.

3.7 Amplification of a virus stock (

see

Note 7)

1. Day 1: Seed T-175 flasks with HeLa cells at 5 × 10

cells per flask. The number of flasks

is determined by the quantity of virus desired.

2. Day 2: Infect each flask, following a standard infection protocol (see following) at

3 pfu/cell. That is, for 10 × 10

cells per flask, infect with 30 × 10

pfu.

Standard infection protocol:

a. Combine equal volumes of virus and trypsin (volume determined by titer of virus

stock). Incubate 37°C for 30 min, vortex every 5 min.

b. Add 5.0 mL of 2.5% DMEM to the virus mixture.

c. Remove media from flask and add the 5.0 mL of virus-media mix.

d. Incubate 2 h at 37°C, with gentle rocking every 15 min.

e. Overlay with 25 mL 2.5% DMEM, and incubate 2–3 d.

3. Scrape cells into 50-mL conical tube and centrifuge at 300g (3000 rpm) for 10 min. Dis-

card the supernatant and resuspend the cell pellet in 2.0 mL of 2.5% DMEM.

4. Rapidly freeze and thaw plaques three times in a Dry Ice/ethanol bath. Store at –80°C

if required.

5. Do a virus titration (see Subheading 3.5.), and aliquot as appropriate.

3.8. Screening for Positive Recombinants by Protein Expression

(

see

Note 8)

1. Day 1: set up cells. Seed 6-well plate with cells at 1 × 10

cells per well. Use 2 wells per

recombinant screen (i.e., perform in duplicate).

2. Day 2: Co-infection should be done with 10–20 pfu/cell for each virus; infection at doses

outside this range will result in poor to no detectable expression. Control cultures should

include (a) uninfected and (b) infected with vTF7-3 alone (at 20–40 pfu/cell to maintain

constant virus load). Also, for UMR-106 or HT-1080 cells, cell density can be assumed to

be 3 × 10

cells per plate at the time of infection. For other cell lines, this needs to be

determined.

3. Thaw recombinant viruses on ice, and add an aliquot to an equal volume of trypsin.

Incubate at 37°C for 30 min, vortexing every 5 min.

4. Mix an appropriate amount of each virus (i.e., titered recombinant and vTF7-3) into

0.5 mL of 2.5% DMEM for each well to be infected.

Recombinant Proteoglycan Expression 215

5. Remove media from cells and wash cell layer twice with PBS.

6. Overlay cells with virus–DMEM cocktail and incubate at 37°C for 2 h, with gentle rock-

ing every 15 min.

7. Add 2 mL of 2.5% DMEM, and incubate a further 4–6 h. This “recovery” period gener-

ally results in a higher rate of protein production, possibly because it allows the sells to

recover somewhat from the conditions of minimal media, as well as allowing time for

viral-related events to occur in the host cell. The optimal rate of protein secretion is gen-

erally seen at 12–16 h postinfection.

8. Biosynthetically label proteins by incubating in the presence of 20–100 µCi/well of

S-Trans label in a final volume of 400 µl of methionine- and cysteine-free DMEM,

serum free (see Note 9).

9. Incubate cells overnight, up to 30 h postinfection.

10. Harvest conditioned media and process by metal chelating chromatography (see

Subheading 3.9.). Due to the nature of the fusion protein cassette, the recombinant

protein should be secreted. However, particularly in the case of mutants, it is possible

that the target protein will not escape quality control systems in the cell. This can be

tested by harvesting the cell layer with a detergent-containing solvent (e.g., 20 mM

Tris-HCl, pH 8.0, 20 mM imidazole, 1% Triton X-100), followed by purification as

described above.

3.9 Metal Chelating Affinity Chromatography (Ni

Column)

This section brifely outlines one strategy for purifying a hexa-histidine-tagged pro-

tein by metal-chelating affinity chromatography. Many similar methods are described

elsewhere, in addition to a number of “kits” and different resins available to achieve

the same goal (see Note 10).

3.9.1. Column Preparation

1. Transfer the desired amount of slurry (for screening, a 0.5-mL resin volume is ample) to

a polypropylene column or, alternatively, use a prepacked column (Amersham Pharmacia

Biotech).

2. Wash resin with ddH

O (5 vol).

3. Charge resin 1× charge buffer (5 vol).

4. Wash resin 1× loading buffer (3 vol).

3.9.2 Sample Preparation

1. Harvest the supernatant from 6-well plates.

2. De-salt the sample on Sephadex G-50 (or PD-10 columns, Pharmacia) column to remove

unincorporated label and exchange sample into load buffer.

Disposable G-50 column preparation:

a. Prepare G-50 slurry in water.

b. Generate a column in a disposable 10-mL pipet, with the end plugged with glass

wool.

c. Wash resin with 3 column volumes of load buffer.

d. Behavior of macromolecular elution is essentially linear up to 20 mL of resin.

i. For 8-mL column, load 2-mL sample and wash in with 0.5 mL, and elute macro-

molecular fraction with 2.5 mL.

ii. For 20-mL column, load 5-mL sample and wash in with 1.25 mL, and elute macro-

molecular fraction with 6.25 mL.

216 McQuillan et al.

3.9.3. Column Chromatography

1. Apply the exchanged sample to the Ni

-charged column.

2. Wash the column with 5 vol of loading buffer.

3. Wash the column with 5 vol of wash buffer.

4. Elute weakly bound material with 3 vol of elution #1.

5. Elute tightly bound proteins with 3 vol of elution #2.

3.9.4. Concentration of Sample and SDS-PAGE

1. Concentrate about one-quarter of each eluted fractions with 10,000-MW cutoff mem-

brane (Centricon, Millipore).

2. Wash concentrate one time with excess TE buffer (10 mM Tris-HCl, 10 mM EDTA), and

reduce volume to ≤ 20 mL.

3. Add SDS-PAGE sample buffer and process as usual for SDS-PAGE analysis, followed

by fixation and fluorography.

4. Core protein and/or proteoglycan should be apparent in positive lanes (see Fig. 3).

3.10. Mass Production

The following protocols briefly describe methods for production of larger amounts

of protein (milligrams and higher), and essentially comprise simple scale-up of the

above protocols. The system lends itself to high-level batch production of recombi-

nant proteins, and purification under native conditions.

1. Day 1: Set up cells. Seed roller bottles (or cell factory) with cells at 5 × 10

cells per

bottle (or per layer of the cell factory). Use 200 mL of 10% DMEM per roller (100 mL

per layer).

2. Allow 1–2 d to reach 80–90% confluence.

3. Day 2: Co-infection. Thaw recombinant viruses and add an equal volume of trypsin.

Incubate at 37°C for 30 min, vortexing every 5 min.

4. Mix an appropriate amount of each virus into 10 mL of 2.5% DMEM for each roller to

be infected.

5. Remove media from cells and wash cell layer twice with PBS.

6. Overlay cells with virus–DMEM cocktail and incubate at 37°C for 2 h in roller apparatus.

7. Add 50 mL of 2.5% DMEM for each roller, and incubate a further 2–4 h.

8. Optional: Biosynthetically label one bottle to use as a trace during purification, by incu-

bating in the presence of 1 mCi per roller of

S-Trans label in a final volume of 30 mL of

methionine- and cysteine-free DMEM. For nonlabeled bottles, use 30 mL of DMEM SF

per roller.

9. Incubate cells overnight, up to 30 h postinfection.

10. Harvest conditioned media and process by metal-chelating chromatography

11. Harvest the supernatant from the roller bottles and add solid imidazole up to a final con-

centration of 5 mM.

11. Add a detergent to reduce nonspecific binding (concentration should be the same as for

the column buffer).

12. Adjust pH to 8.0 using concentrated NaOH.

13. Purify hexa-histidine tagged proteoglycan by affinity purification on Ni

-charged

metal-chelating column (see Subheading 3.9.), using imidazole gradient elution as

shown in Fig. 3.

Recombinant Proteoglycan Expression 217

3.11. Characterization of Recombinant Proteoglycan

Following purification of recombinant proteoglycan and core protein by the

procedures described above, further steps are dictated by the ultimate disposition of

the protein. Simple carbohydrate analysis should yield information about glycosami-

noglycan type, number, and size; more detailed analysis will show substitution with

N- and O-linked oligosaccharides; and finally, study of the structure (e.g., CD spec-

troscopy, NMR analysis, crystallization) and biological function (e.g., interaction with

other matrix components, role in ECM assembly, in-vivo activity) will take advantage

of the high-level expression and purification of native proteoglycan that is likely to

closely resemble the in-vivo product. Many of these methods are detailed elsewhere in

this book.

4. Notes

1. Constructs developed for expression in the vaccinia/T7 phage hybrid system have a T7

promoter driving expression of the target protein. Following construction of an expres-

Fig. 3. Purification of recombinant proteoglycan by metal-chelating affinity chromatogra-

phy. (a)

S-labeled proteoglycan (decorin) in conditioned media was applied to a Ni

-charged

iminodiacetic acid column (2 mL) in 20 mM imidazole buffer. Bound material was eluted by a

stepwise increase of imidazole to 60 mM (proteoglycan elutes), and a linear gradient of imida-

zole from 60 to 200 mM (core protein elutes); see text for discussion. (b) Analysis of selected

fractions (indicated by the asterisk in panel a) by SDS-PAGE and visualized by fluorography.

Note that the separation of proteoglycan and core protein glycoforms is likely to be specific to

decorin (and biglycan) due to the proximity of the glycosaminoglycan chain to the hexa-histi-

dine tag resulting in a lower affinity interaction.

218 McQuillan et al.

sion vector in any of the pT-cam series of vectors, we recommend doing in-vitro tran-

scription directly from the plasmid vector with T7 RNA polymerase, and in-vitro transla-

tion with a reticulocyte lysate system. This is in addition to direct sequencing through

cloning sites or, optimally, through the entire coding region. A significant amount of

wasted time can be avoided by following these two simple steps.

2. Since there is likely to be background wild-type virus as well as spontaneous deletion of

the TK gene activity, it is recommended to take 8 plaques through to the second round.

Some of these may not survive a second round of selection, and others may not express

protein. Ideally, 50–75% of the first round plaques should be the desired recombinant;

however, this efficiency can be somewhat less (ultimately, you need only one positive!).

3. If pg 1.1.3 is a positive, the name indicates it is derived from plaque number pg 1 from the

pg recombination, and that it has gone through three rounds of selection. If pg 1.1.3 is lost

through a laboratory accident, then it is likely that pg 1.1.1, pg 1.1.2, and pg 1.1.4 are

identical clones and the positive can be recovered.

4. It is possible to screen plaques at this stage by PCR analysis. Our experience suggests that

it is difficult to design appropriate positive and negative controls for this procedure. Since

the ultimate test of a recombinant virus will always be expression of the protein of inter-

est, we recommend amplifying the eight recombinants, determining the titration, and

screening for protein expression. However, PCR screening can be useful, particularly

when generating and screening a large number of constructs (truncations, deletions,

mutations, etc.). Therefore this protocol describes the isolation of viral DNA for subse-

quent PCR by standard methods using appropriately designed oligonucleotides.

5. These procedures are followed to generate enough virus for an accurate titration and to

screen for protein expression. We recommend amplifying 4–8 recombinants from the

third-round plaque assay.

6. An accurate titration is required before any attempts to screen for protein expression.

Titers can vary by 1-2 orders of magnitude, and optimal protein expression is achieved

only within a discrete range of infection (5–30 pfu/cell). Infection with too little or too

much virus will not yield detectable levels of protein expression.

7. Following identification of a positive recombinant, large stocks of virus are made and can

be aliquoted and stored at –70°C for extended periods. For generation of milligram

amounts of protein, and to avoid significant batch-to-batch variation, it is recommended

to make as much virus stock as is practical.

8. After generating up to eight putative positive recombinant viruses that have undergone at

least three rounds of selection in the presence of BrdU, a simple proteoglycan/protein

expression screen is done to identify genuine positives. There are a number of ways to

screen for the protein of interest, including Western blotting with specific antibodies or

even sensitive bioassays if available. However, in our laboratory we almost exclusively

utilize biosynthetic labeling of the core protein and purification with the hexa-histidine

tag, followed by predicted migration on SDS-PAGE.

9. Most cells will tolerate serum-free conditions for the period of labeling, and this has the

advantage of reducing total protein in the harvested conditioned medium. However, for

cell lines that are particularly sensitive to serum-free conditions, one can add minimal

serum or some other form of defined medium.

10. The behavior of a particular protein is not easily predicted, and better performance can

often be achieved by an empirical approach, testing different resins, different divalent

cautions, and changes in solvent conditions. However, for the purposes of an initial screen,

this method is appropriate.

Recombinant Proteoglycan Expression 219

References

1. Chan, D., Lamande, S. R., McQuillan, D. J., and Bateman, J. F. 1997. In vitro expression

analysis of collagen biosynthesis and assembly. J. Biochem. Biophys. Meth. 36, 11–29.

2. Chan, D., Weng, Y. M., Hocking, A. M., Golub, S., McQuillan, D. J. and Bateman, J.

F. 1996. Site-directed mutagenesis of human type X collagen. Expression of alpha1X)

NC1, NC2, and helical mutations in vitro and in transfected cells. J. Biol. Chem. 271,

13,566–13,572.

3. Hocking, A. M., Strugnell, R., Ramamurthy, A. P., and McQuillan, D. J. 1996. Eukaryotic

expression of recombinant biglycan. Post-translational processing and the importance of

secondary structure for biological activity. J. Biol. Chem. 271, 19,571–19,577.

4. Krishnan, P., Hocking, A. M., Scholtz, J. M., Pace, C. N., Holik, K. K., and McQuillan,

D. J. 1999. Distinct secondary structures of the leucine-rich repeat proteoglycans decorin

and biglycan. Glycosylation-dependent conformational stability. J. Biol. Chem.

274,10,945–10,950.

5. Ramamurthy, P., Hocking, A. M., and McQuillan, D. J. 1996. Recombinant decorin

glycoforms. Purification and structure. J. Biol. Chem. 271, 19,578–19,584.

6. Hering, T. M., Kollar, J., Huynh, T. D., and Varelas, J. B. 1996. Purification and

characterization of decorin core protein expressed in Escherichia coli as a maltose-binding

protein fusion. Anal. Biochem. 240, 98–108.

7. Hildebrand, A., Romaris, M., Rasmussen, L. M., Heinegard, D., Twardzik, D. R., Border,

W. A., and Ruoslahti, E. 1994. Interaction of the small interstitial proteoglycans biglycan,

decorin and fibromodulin with transforming growth factor beta. Biochem. J. 302, 527–534.

8. Schonherr, E., Hausser, H., Beavan, L., and Kresse, H. 1995. Decorin-type I collagen inter-

action. Presence of separate core protein-binding domains. J. Biol. Chem. 270, 8877–8883.

9. Schonherr, E., Witsch-Prehm, P., Harrach, B., Robenek, H., Rauterberg, J., and Kresse, H.

1995. Interaction of biglycan with type I collagen. J. Biol. Chem. 270, 2776–2783.

10. Groffen, A. J., Buskens, C. A., Tryggvason, K., Veerkamp, J. H., Monnens, L. A., and van

den Heuvel, L. P. 1996. Expression and characterization of human perlecan domains I and

II synthesized by baculovirus-infected insect cells. Eur. J. Biochem. 241, 827–834.

11. Gu, J., Nakayama, Y., Nagai, K., and Wada, Y. 1997. The production and purification of

functional decorin in a baculovirus system. Biochem. Biophys. Res. Commun. 232, 91–95.

12. Hochuli, E., Dobeli, H., and Schacher, A. 1987. New metal chelate adsorbent selective for pro-

teins and peptides containing neighbouring histidine residues. J. Chromatogr. 411, 177–184.

13. Mackett, M., Smith, G. L., and Moss, B. 1985. The construction and characterization of

vaccinia virus recombinants expressing foreign genes; in DNA Cloning, volume II (Glover,

D. M., ed.), IRL, Oxford, UK, pp.191–212.

14. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. M., Seidman, J. G., Smith, J. A., and

Struhl, K., eds. 1987. Current Protocols in Molecular Biology. Wiley, New York, NY.

15. Elroy-Stein, O., Fuerst, T. R., and Moss, B. 1989. Cap-independent translation of mRNA

conferred by encephalomyocarditis virus 5' sequence improves the performance of the

vaccinia virus/bacteriophage T7 hybrid expression system. Proc. Natl. Acad. Sci. (USA)

86, 6126–6130.

16. Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B. 1986. Eukaryotic transient-expres-

sion system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA

polymerase. Proc. Natl. Acad. Sci. (USA) 83, 8122–8126.

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polymerase system for expression of target genes. Mol. Cell. Biol. 7, 2538–2544.

Purification of Recombinant Decorin 221

221

From:

Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols

Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Purification of Recombinant Human Decorin

and Its Subdomains

Anna L. McBain and David M. Mann

1. Introduction

Conventional approaches to purifying most connective tissue proteoglycans (PGs)

generally involve treatments that consequently alter the binding properties of the

purified PG (e.g., exposure to strong detergents or harsh denaturants such as

guanidinium). Herein we describe a protocol that we use to isolate human decorin in

which the PG remains functional in its binding to various ligands (1,2). This method

utilizes a eukaryotic expression system that produces high quantities of recombinant

human decorin as a soluble component of the transfected cell culture medium together

with a combination of conventional ion-exchange and hydrophobic-interaction chro-

matography. Here we provide detailed instructions for (1) the production of condi-

tioned medium containing secreted recombinant human decorin, (2) the initial

ion-exchange chromatography step using DEAE-Sepharose, and (3) the final hydro-

phobic-interaction chromatography step.

In addition, we have also developed a prokaryotic system for the expression of

subdomains of human decorin. This system utilizes a derivative of the pATH plasmid

series (3) and was selected because our preliminary studies indicated that it yields high

levels of recombinant decorin core protein with minimal degradation. Decorin

subdomains are expressed in Escherichia coli as fusion hybrids with the amino-terminal

323 residues of anthranilate synthase (trpE), which tends to stabilize the fusion protein

and cause it to form protease-resistant inclusion bodies within the cytosol. Inclusion

body formation also facilitates isolation of the decorin-subdomain fusion proteins.

We used a cassette mutagenesis approach to engineer a derivative of the pATH3

expression vector to introduce several desirable features. For example, our modified

pATH3 vector encodes a Factor Xa cleavage site between the trpE encoded sequence

and the inserted decorin sequence, and a polyhistidine tag at the carboxy-terminus of

the decorin sequence. This tag provides an affinity handle to facilitate a rapid and

highly specific method of purifying the expressed protein from bacterial cell extracts

222 McBain and Mann

if needed. We have used E. coli WM6 as the host cell for expression of the decorin

fusion proteins because these cells are deficient in protease activity (4). They are a

derivative of the E. coli strain CAG 456, which carry a mutation in the RNA

polymerase sigma subunit specific for heat-shock promoters. Thus, in addition to a

reduced proteolysis, there is a potential to accumulate the expressed protein for longer

periods of time in these cells. In this chapter, we describe the general outline for

subcloning a decorin core protein domain into our modified pATH3 vector and provide

a protocol for expressing this subdomain as an anthranilate synthase hybrid protein.

We also have provided our protocol for the isolation and solubilization of the resulting

inclusion bodies.

2. Materials

2.1. Cell Culture and Conditioned Medium Production

1. Chinese hamster ovary cells stably transfected to express the proteoglycan form of human

decorin (5).

2. Supplemented alpha(–)MEM: Alpha(–)MEM (without nucleosides) supplemented with

final concentrations of 25 mM glucose, 0.8 mM sodium sulfate, 0.02% Tween 80 (Sigma,

St. Louis, MO), 100 units/mL penicillin G sodium, 100 units/mL streptomycin sulfate, and

0.292 mg/mL

L-glutamine.

3. Serum-containing supplemented alpha(–)MEM: As described above, with 10% defined and

supplemented bovine calf serum (Hyclone Laboratories, Logan, UT) added.

4. Trypsin EDTA 1× solution in Hanks’ balanced salts (0.05% Trypsin, 0.53 mM EDTA,

without calcium or magnesium).

5. 850 cm

polystyrene roller bottles (Corning, Corning, NY).

6. Cell wash buffer: Dulbecco’s phosphate–buffered saline containing 1 mM calcium chlo-

ride and 0.5 mM magnesium chloride.

7. Protease inhibitor stocks: 1 mg/mL aprotinin, 1 mg/mL leupeptin, 1 mg/mL pepstatin A.

8. Sodium azide stock solution (20% w/v).

2.2. DEAE-Sepharose Chromatography

1. Buffer A: 300 mM NaCl in phosphate buffer, pH 7.4 (6.5mM Na

HPO

.7H

O, 1.1 mM

, 2.7 mM KCl) with 0.02% sodium azide.

2. 5 M NaCl in water.

3. Buffer B: 6 M urea containing 6 mM CHAPS and 300 mM NaCl in phosphate buffer,

pH 7.4 (as defined above), with 0.02% sodium azide. Prepare this urea buffer fresh from

a 9 M urea stock that has been maintained over a mixed-bed ion-exchange resin, TMD-8

(Sigma, St. Louis, MO) to remove cyanate ions.

4. Buffer C: 1 M NaCl in phosphate buffer (as defined above), pH 7.4, with 0.02% sodium

azide.

5. Two 2.5 cm (diameter) by 20 cm (length) chromatography columns (e.g., Kontes Flex),

each packed with 80-mL DEAE-Sepharose Fast Flow (Pharmacia).

6. A 2.5 cm by 20 cm guard column packed with 30-mL Sepharose-4B (Pharmacia).

2.3. Hydrophobic Interaction Chromatography (HIC)

1. Saturated ammonium sulfate (4.1 M at 25°C) in 50 mM phosphate buffer, pH 6.3.

2. A 50 mm by 4.6 mm Hydrocell C4-1000 HIC column, 10 µm by 1000 Å (prepacked by

BioChrom Labs, Terre Haute, IN).

Purification of Recombinant Decorin 223

3. 2.5 M ammonium sulfate in 50 mM phosphate buffer, pH 6.3.

4. 50% Ethylene glycol in 50 mM phosphate buffer, pH 8.3.

2.4. Prokaryotic Expression of Decorin Subdomains

as Anthranilate Synthase Hybrid Proteins

1. pATH3 prokaryotic expression plasmid (American Type Culture Collection [ATCC],

Manassas, VA, product #37697), modified within the multiple cloning site by cassette

mutagenesis to contain the following: Factor Xa protease cleavage site (Ile-Glu-Gly-Arg),

a directional cloning site (BamHI-XhoI-XbaI) for PCR-generated decorin domains, an

acid-labile cleavage site (Asp-Pro), and a polyhistidine affinity tag (six consecutive histi-

dine residues) followed by two consecutive lysine residues that can be used for chemical

cross-linking to a cyanogen bromide-activated agarose matrix.

2. PCR product encoding the desired subdomain of human decorin, terminating with an

appropriate restriction site for subcloning into the expression vector above such that the

decorin codons remain in frame with those of the anthranilate synthase sequence.

3. E. coli WM6 (ATCC product #47020, genotype F- supC(Ts) lac(Am) mal trp(Am) rpsL

htpR165 pho(Am) lambda-).

4. Luria-Bertani medium (LB medium).

5. M9 minimal medium with casamino acids.

6. Ampicillin: 50 mg/mL stock in water.

7. Indoleacrylic acid stock of 10 mg/mL in ethanol (this should be prepared immediately

before use).

8. Phenylmethylsulfonylfluoride (PMSF): 200 mM stock in 95% ethanol.

9. Lysis buffer: 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1 M NaCl.

10. Lysis buffer with 0.5% Triton X-100 and 10 mM EDTA.

11. Lysis buffer with 8 M Urea and 2 mM PMSF.

12. Deoxycholic acid (sodium salt, Sigma).

13. Deoxyribonuclease I (DNaseI) from bovine pancreas (Sigma), dissolved at 1 mg/mL in

Tris-buffered saline.

3. Methods

3.1. Cell Culture and Conditioned Medium Production

1. Chinese hamster ovary cells stably transfected with the proteoglycan form of human

decorin are grown in serum-containing alpha(–)MEM medium at 37°C in 5% CO

. We

typically grow these cells in 75 cm

tissue culture flasks until confluent and then expand

them into 850 cm

roller bottles. After detaching cells from one confluent 75 cm

flask

using trypsin/EDTA, the cells are gently pelleted by centrifugation at 320g, 25°C. The

trypsin/EDTA supernatant is discarded and the resuspended cell pellet is added to

150 mL of serum-containing medium in an 850 cm

roller bottle. Cells are maintained in

serum-containing supplemented alpha(–)MEM until they have reached confluency. Roller

bottle cultures should be grown at 37°C in 5% CO

with a rotational speed of 0.5 rpm.

2. Remove the serum-containing conditioned medium (see Note 1) and wash the adherent

cell layer three times each with approximately 150 mL of cell wash buffer at 37°C.

3. Add 50 mL of supplemented alpha(–)MEM (serum-free) to the roller bottle and rotate the

cells in this medium at 0.5 rpm and 37°C for 8–24 h to allow them to condition this

medium with secreted decorin (see Note 2).

4. Collect the serum-free conditioned medium and refeed cells with 150 mL of serum-con-

taining medium. Culture cells in this medium for at least 48 h to allow them to recover

from the serum deprivation before producing serum-free conditioned medium again.

224 McBain and Mann

5. Add protease inhibitors (aprotinin, leupeptin, pepstatin A) to the conditioned medium to

a final concentration of 1 µg/mL for each (see Note 3).

6. Sodium azide should be added to the conditioned medium to a final concentration of

0.02% to prevent microbial contamination during subsequent handling steps.

7. Remove cell debris by centrifugation for 10 min at 430g and store clarified conditioned

medium at –80°C (preferred) or –20°C.

8. Cultures maintained in roller bottles should be harvested and reseeded into fresh roller bottles

at approximately 1 × 10

cells per roller bottle every 2–3 wk or whenever significant slough-

ing can be observed macroscopically (i.e., greater than 20% of the surface area is cell free).

Production of conditioned medium from roller bottle cultures that are overgrown will result in

increased histone and DNA contamination of the decorin due to significant cell lysis.

3.2. DEAE-Sepharose Chromatography

1. Thaw conditioned medium at 37°C.

2. Bring 20–30 L of pooled medium to a final concentration of 300 mM NaCl by adding 31 mL

of 5 M NaCl stock per liter of conditioned medium (see Notes 4 and 5).

3. Apply the conditioned medium over two 80-mL DEAE-Sepharose fast flow columns con-

nected and preceded by a 30-mL Sepharose-4B precolumn guard to remove nonspecific

Sepharose-binding molecules (columns should be previously equilibrated with buffer A).

This should be performed at 4°C with a flow rate not exceeding 3 mL/min. Typically we pass

15 L over these columns in about 72 h using gravity flow without pumping (see Note 6).

4. Detach the preguard column and individually wash the 80 mL DEAE-Sepharose columns

each with 2–3 L of buffer A until the absorbance at 280 nm of this wash is below 0.010.

This step can be performed at room temperature with a gravity-driven flow rate of

approx 750 mL/h (see Note 7).

5. Wash each DEAE-Sepharose column seperately with 250 mL of buffer B. The presence of

6 mM CHAPS in this buffer removes low-molecular-weight decorin-binding contaminants (6).

6. Remove the urea/CHAPS thoroughly by washing each column with approx 0.5–1.0 L of

buffer A.

7. Place the two DEAE-Sepharose columns back in series and remove the bound decorin

with a linear NaCl gradient generated between 100% buffer A and 100% buffer C (300 mM

to 1 M NaCl, in phosphate buffer, pH 7.4). We recommend performing this step at 4°C

and pumping a total elution volume of 500 mL at a constant flow rate of 20–30 mL/h while

collecting 50 × 10 mL fractions.

8. Measure the absorbance of each fraction at 280 and 260 nm and plot these values vs the

fraction number. Typically, the elution profile will contain two partially overlapping peaks,

the first composed predominately of nucleic acid contaminants and the second, smaller, peak

containing recombinant human decorin. The bound decorin will dissociate from the DEAE

columns in approx 600–800 mM NaCl.

9. Analyze the purity of decorin in the fractions by SDS-PAGE (see Note 8). Pool the

decorin-containing fractions that lack significant DNA contamination (determined

by absorbance at 260 nm). Our typical yields of recombinant human decorin are

approx 2.5–3.0 mg/L from serum-free conditioned medium (see Note 9). Greater yields

(5–10 mg/L) are obtained from serum-containing medium that has been conditioned for

48 h or longer.

3.3. Hydrophobic Interaction Chromatography

1. Dilute the pooled decorin-containing fractions with an equal volume of saturated

ammonium sulfate in 50 mM phosphate buffer, pH 6.3.