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 conditioned 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

From: Methods in Molecular Biology, Vol. 171:Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

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 1x solution in Hanks' balanced salts (0.05% Trypsin, 0.53 mM EDTA, without calcium or magnesium).

5. 850 cm2 polystyrene roller bottles (Corning, Corning, NY).

6. Cell wash buffer: Dulbecco's phosphate-buffered saline containing 1 mM calcium chloride 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 Na2HPO4.7H2O, 1.1 mM KH2PO4, 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 ^m by 1000 A (prepacked by BioChrom Labs, Terre Haute, IN).

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-Xhol-Xbal) 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 htpR165pho(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 (DNasel) 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% CO2. We typically grow these cells in 75 cm2 tissue culture flasks until confluent and then expand them into 850 cm2 roller bottles. After detaching cells from one confluent 75 cm2 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 cm2 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% CO2 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-containing 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.

5. Add protease inhibitors (aprotinin, leupeptin, pepstatin A) to the conditioned medium to a final concentration of 1 ^g/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 x 107 cells per roller bottle every 2-3 wk or whenever significant sloughing 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 connected 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 x 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.

Fig. 1. Purification of human recombinant decorin by hydrophobic interaction chromatography. (A) Representative chromatogram resulting from C4-hydrophobic interaction column loaded with pooled DEAE-Sepharose purified decorin. Note the three predominant peaks detected in the eluted material, P1-P3. (B) SDS-PAGE analysis of three peaks fractionated by HIC: lane 1, Coomassie blue toluidine blue staining of the starting material loaded on the HIC column; lanes 2-5, immunoblot analysis of the starting material (lane 2) and the eluted peaks, P1-P3 (lanes 3-5, respectively), using a polyclonal antibody directed against the amimo terminal end of human decorin (7).

Fig. 1. Purification of human recombinant decorin by hydrophobic interaction chromatography. (A) Representative chromatogram resulting from C4-hydrophobic interaction column loaded with pooled DEAE-Sepharose purified decorin. Note the three predominant peaks detected in the eluted material, P1-P3. (B) SDS-PAGE analysis of three peaks fractionated by HIC: lane 1, Coomassie blue toluidine blue staining of the starting material loaded on the HIC column; lanes 2-5, immunoblot analysis of the starting material (lane 2) and the eluted peaks, P1-P3 (lanes 3-5, respectively), using a polyclonal antibody directed against the amimo terminal end of human decorin (7).

2. Apply this material to a C4 hydrophobic interaction column (previously equilibrated with 2.5 M ammonium sulfate in 50 mM phosphate buffer, pH 6.3) at room temperature with a slow flow rate (e.g., 1.0 mL/min for a 50 by 4.6 mm column) and wash the column with the equilibration buffer until a baseline signal is achieved (see Note 10).

3. Remove bound decorin with a reverse linear gradient of ammonium sulfate from 2.5 to 0 M in 50 mM phosphate buffer, pH 6.3, while simultaneously applying an increasing linear gradient of ethylene glycol from 0 to 50% in 50 mM phosphate buffer, pH 8.3. When monitored by absorbance at 280 nm, we typically observe three major peaks eluting from the column (Fig. 1A; see Note 11). Coomassie blue/toluidine blue-staining SDS-polyacryla-mide gels and immunoblotting using a polyclonal antibody directed against the amino-ter-minal end of human decorin (7) reveal that the second and third peaks in the chromatogram contain highly purified decorin, with no other molecules detected (Fig. 1B).

4. Collect decorin containing peaks and remove ethylene glycol and ammonium sulfate by dialysis using a 30-kDa MWCO dialysis membrane (Spectrapor). We typically dialyze against phosphate-buffered saline before storing the final decorin product.

3.4. Prokaryotic Expression of Decorin Sequences as Anthranilate Synthase Fusion Proteins

1. Cloning of human decorin subdomains into modified pATH3 vector: PCR amplify the desired decorin core protein sequence(s) using synthetic oligonucleotide primers that are designed to incorporate appropriate restriction sites for subcloning the product into the modified pATH3 vector in the correct reading frame. Restrict PCR product with appropriate restriction enzymes (e.g., BamHI and Xbal) and subclone into the modified pATH3 vector. Figure 2 illustrates the boundaries of the decorin subdomains that we have subcloned into our modified pATH3 vector and expressed as fusion proteins, as well as the sequence of the salient features of this expression vector and resultant fusion polypeptides.

Proteoglycan Structure

Fig. 2. Structure of decorin core protein and prokaryotic expression vector used to produce anthranilate synthase-decorin hybrid proteins. (A) Delineation of human decorin subdomains that have been expressed as hybrid proteins. (B) Model of hybrid fusion protein illustrating the salient features introduced by expression from our modified pATH3 vector. (C) Sequence of the fusion site in modified pATH3 vector illustrating position of specific engineered features.

Fig. 2. Structure of decorin core protein and prokaryotic expression vector used to produce anthranilate synthase-decorin hybrid proteins. (A) Delineation of human decorin subdomains that have been expressed as hybrid proteins. (B) Model of hybrid fusion protein illustrating the salient features introduced by expression from our modified pATH3 vector. (C) Sequence of the fusion site in modified pATH3 vector illustrating position of specific engineered features.

2. Expression of fusion proteins in E. coli WM6: Use E. coli WM6 cells to express the recombinant fusion protein. Prepare a freshly seeded plate with WM6 transformants and pick a single colony for overnight expansion in LB medium containing 100 ^g/mL ampi-cillin (37°C while shaking at 225 rpm). Dilute the overnight culture with 4 volumes of M9 minimal medium containing casamino acids supplemented with 100 ^g/mL ampicil-lin (i.e., cell density diluted to 20% that of the overnight culture) (see Note 12). Incubate the diluted culture for 3 h at 37°C with shaking and then induce fusion protein expression by adding indoylacrylic acid (IAA) to a final concentration of 10 ^g/mL. Incubate cultures in IAA containing medium for 5 h at 37°C with shaking at 225 rpm to accumulate high levels of expressed fusion protein (see Note 13). Add PMSF to the cultures to a final concentration of 0.2 mM. At this stage the total bacterial culture can be analyzed for the expression of the fusion protein. Remove 10 ^L of culture, dilute with 2x SDS-PAGE sample buffer (reducing), and analyze total protein content by SDS-PAGE (see Note 14). Figure 3 shows the profile of total proteins present in the WM6 cultures (cell + medium) and their immunoreactivity with rabbit polyclonal antisera raised against either human decorin (7) or a carboxy-terminal peptide of human decorin (8).

Fig. 3. Expression of anthranilate synthase-decorin hybrid proteins in E. coli WM6. Fractionation of 5-20% SDS-polyacrylamide gel of total proteins present in combined media and cell pellet: (A) Ponceau S staining of a nitrocellulose membrane before immunodetection; (B,C) immunoblot analysis using rabbit polyclonal antisera raised angainst either human decorin proteoglycan or a carboxy-terminal peptide of the core protein of human decorin (8), respectively. Total proteins derived from WM6 cultures transformed with (lane 1) modified pATH3 vector only (no decorin sequence inserted); (lane 2) modified pATH3 vector with decorin NH2 domain insert (Asp1-Lys44); (lane 3) modified pATH3 vector with decorin LRM domain insert (Val45-Gly278); (lane 4) modified pATH3 vector with decorin COOH domain insert (Ser279-Lys329); (lane 5) modified pATH3 vector with whole decorin sequence insert (Asp1-Lys329). As a positive control, lane D contains the proteoglycan from of human decorin.

Fig. 3. Expression of anthranilate synthase-decorin hybrid proteins in E. coli WM6. Fractionation of 5-20% SDS-polyacrylamide gel of total proteins present in combined media and cell pellet: (A) Ponceau S staining of a nitrocellulose membrane before immunodetection; (B,C) immunoblot analysis using rabbit polyclonal antisera raised angainst either human decorin proteoglycan or a carboxy-terminal peptide of the core protein of human decorin (8), respectively. Total proteins derived from WM6 cultures transformed with (lane 1) modified pATH3 vector only (no decorin sequence inserted); (lane 2) modified pATH3 vector with decorin NH2 domain insert (Asp1-Lys44); (lane 3) modified pATH3 vector with decorin LRM domain insert (Val45-Gly278); (lane 4) modified pATH3 vector with decorin COOH domain insert (Ser279-Lys329); (lane 5) modified pATH3 vector with whole decorin sequence insert (Asp1-Lys329). As a positive control, lane D contains the proteoglycan from of human decorin.

3. Isolation and solubilization of fusion protein inclusion bodies: Pellet WM6 cells by cen-trifugation at 5000g for 15 min at 4°C and determine the weight of the cell pellet. Resuspend the cell pellet in lysis buffer using 3 mL of lysis buffer per gram of wet cell pellet weight. While shaking cell suspension at 25 °C, add 4 mg of deoxycholic acid per gram of original cell pellet weight. Incubate lysate at 37°C until it becomes viscous (10-30 min) and then add 20 ^L of DNase I solution per gram cell pellet weight. Incubate at 25 °C until no longer viscous (30-60 min). Centrifuge at 12,000g for 15 min at 4°C. Decant supernatant and resuspend pellet in 9 vol of lysis buffer containing 0.5% Triton X-100 and 10 mM EDTA, pH 8.0. Incubate for 5 min at 25°C before centrifuging for 15 min at 12,000g, 4°C. Decant supernatant and solubilize the residual pellet in lysis buffer containing 8 M urea and 2 mM PMSF. Analyze all supernatants and urea insoluble pellet by SDS-PAGE to determine the distribution of the fusion protein.

4. Notes

1. Serum-containing medium can be processed as per steps 5-7 under Subheading 3.1., but, the decorin produced from this material can contain low levels of bovine proteoglycans derived from the serum. By collecting and processing conditioned medium made under serum-free conditions, the levels of contaminating proteoglycans from the bovine serum are undetectable.

2. We have found that 16 h is an optimal period to allow cultures to condition their serumfree medium with decorin. Longer incubations tend to increase DNA contamination in the decorin preparations, due to increased cell lysis, while shorter incubations yield lower concentrations of decorin.

3. It is important to add protease inhibitors before the centrifugation of conditioned medium in order to reduce degradation of decorin core protein caused by proteases released from any lysed cells.

4. Increasing the ionic strength of the conditioned medium prior to performing ion-exchange chromatography reduces binding of weak polyanionic molecules to the DEAE-Sepharose matrix and thus increases the binding capacity of the column and the purity of the isolated decorin.

5. For handling of large volumes of conditioned medium we typically use the 5-gal plastic bottles from common water coolers (each will hold 18-19 L of conditioned medium).

6. Occasionally it may be necessary to siphon off the top 1-2 mm of the matrix from the guard column, as this may become clogged with a floculant precipitate.

7. To reduce loss of decorin in the wash buffer of the DEAE-Sepharose column, the total wash time should not exceed 4 h. The fast flow rates necessary can be achieved under a gravity-driven flow by positioning the connected wash bottle approximately 7 ft (2 m) above the column.

8. We typically analyze the purity of eukaryotic expressed decorin during the purification scheme by electrophoresing samples under reducing conditions on 5-20% SDS-polyacry-lamide gradient gels. These gels are subsequently stained with Coomassie blue to detect any protein contaminants and then with 0.1% toluidine blue (w/v) in 0.1 M acetic acid, to detect proteoglycans.

9. The DEAE column flow-through conditioned medium can be reapplied to the ion-exchange columns for an approx 10% additional yield of decorin.

10. We use the Hydrocell C4-1000, 50 mm x 4.6 mm (BioChrom Labs) on a TOSOH HPLC system (TSK 6011) equipped with a TSK 6041 UV detector and a GM8010 gradient monitor; however, a Pharmacia FPLC system (or other equivalent) can be used.

11. Preliminary preparative runs using the larger, 150 mm x 21 mm, Hydrocell C4-1000 column indicate that approx 600 mg of recombinant human decorin can be bound and recovered from this column.

12. Although the pATH3 expression vector uses a fairly tight promoter, if low yields of the fusion protein are observed expression can sometimes be improved by depleting tryptophan levels in the cultures for 2-3 h before inducing expression with IAA. For example, the overnight LB cultures are separated from the tryptophan-containing spent medium by cen-trifugation and resuspended at 20%, their overnight density in tryptophan-free M9 medium containing casamino acids.

13. The optimal expression period must be determined emperically for each particular fusion molecule. For example, depending on the stability and accumulation of individual proteins, the induction period can range from 2 to 12 h.

14. To determine whether the induction step has been successful, it is advised that a duplicate culture not induced by IAA be prepared and analyzed by SDS-PAGE.

References

1. Moscatello, D. K., Santra, M., Mann, D. M., McQuillan, D. J., Wong, A. J., and Iozzo, R. V.

(1998) Decorin suppresses tumor cell growth by activating the EGF-receptor. J. Clin. Invest.

2. Yamaguchi, Y., Mann, D. M., and Ruoslahti, E. (1990) Transforming growth factor is negatively regulated by a proteoglycan. Nature 346, 281-284.

3. Koerner, T. J., Hill, J. E., Myers, A. M., and Tzagoloff, A. (1991) High-expression vectors with multiples cloning sites for construction of trpE fusion genes: pATH vectors. Meth. Enzymol. 94, 477-490.

4. Mandecki, W., Powell, B. S., Mollison, K. W., Carter, G. W., and Fox, J. L. (1986) Highlevel expression of a gene encoding the human complement factor C5a in Escherichia coli. Gene 43, 131-138.

5. Yamaguchi, Y. and Ruoslahti, E. (1988) Expression of human proteoglycan in Chinese hamster ovary cells inhibits cell proliferation. Nature 336, 244-246.

6. Choi, H. U., Johnson, T. L., Pal, S., Tang, L-H, Rosenberg, L., and Neame, P. J. (1989) Characterization of the dermatan sulfate proteoglycans, DS-PGI and DS-PGII, from bovine articular cartilage and skin isolated by octyl-sepharose chromatography. J. Biol. Chem.

264, 2876-2884.

7. Mann, D. M., Yamaguchi, Y., Bourdon, M. A., and Ruoslahti, E. (1990) Analysis of glycosaminoglycan substitution in decorin by site-directed mutagenesis. J. Biol. Chem.

265, 5317-5323.

8. Roughly, P. J., White, R. J., Magny, M-C., Liu, J., Pearce, R. H., and Mort, J. S. (1993) Non-proteoglycan forms of biglycan increase with age in human articular cartilage. Biochem. J. 295, 421-426.

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