Construct Assembly of Target Proteoglycans

1. Target proteoglycan gene.

2. Specifically designed oligonucleotides with certain features, e.g., restriction sites, His6 tag, etc., as desired.

3. PCR-Script SK(+) cloning vector kit (Stratagene).

4. Escherichia coli (E. coli ) XL-blue MRF' Kan supercompetent cells.

5. X-gal (5-bromo-4-chloro-3-indolyl p-D-galactopyranoside), IPTG (isopropyl p-D-thio-galactopyranoside), and N,N-dimethyl formamide (Sigma).

6. Restriction enzymes and matching buffers corresponding to the engineered restriction sites.

7. DNA purification system Minipreps (Wizard Plus from Promega).

8. LB broth base (Life Technology).

9. Eukaryotic expression vector (e.g., pcDNA3 from Invitrogen). 10. T4 DNA ligase (Invitrogen).

II. E. coli TOPIOF' competent cells (Invitrogen).

12. Ampicillin-containing agar plates (only if ampicillin is the selecting reagent).

13. QiaFilter DNA purification systems (for Maxipreps from Qiagen).

14. Sequencing oligonucleotides (pairing to expression vector or cloning vector or templates).

15. Agarose (Life Technology).

16. NanoSep 0.2-^m DNA filters (Fisher Scientific/Pall Gelman).

17. 125-mL glass flask.

18. 15-mL Falcon tube.

19. DNA sequence verification software (optional).

2.2. Expression of Target Proteoglycans

1. Eukaryotic cell line(s) (e.g., Chinese Hamster Ovary K1 cells, COS cells).

2. Lipofectamine + PLUS reagent (Life Technology).

3. Regular minimal essential culture medium (MEM), regular OPTI-MEM (Life Technology), and methionine-free and cysteine-free OPTI-MEM (Life Technology, special order).

4. Fetal bovine serum (certified grade).

5. Antibiotic-antimycotic solution (containing100 U/mL penicillin, 100 ^g/mL streptomycin, 250 ng/mL amphotericin, Life Technology).

6. Radioactive isotopes 35S-Pro-Mix (methionine and cysteine, Amersham, 7.15 mCi, for 4-5 plates of cells) for methionine + cysteine labeling (labeling core proteins) or 35S-Na2SO4 (sodium sulfate, Dupont NEN, 5 mCi, for 4-5 plates of cells) for sulfate labeling (labeling sulfated glycosaminoglycan chains).

7. Decolorizing NORIT-A carbon (ACROS Organics).

8. Sterilized 9-in. glass pipets (Fisher Scientific).

9. 10-mL individually wrapped plastic pipets (sterilized).

10. Cell culture dishes (100 mm).

11. Eppendorf tubes (regular and boiling tubes).

12. Table-top centrifuge (up to 14,000g speed).

13. Nonidet P-40 detergent.

14. Phenylmethylsulfonyl fluoride (PMSF, see Subheading 3.2.3.), leupeptin, antipain, benzamidine, aprotinin, chymostatin, pepstatin, dimethyl sulfoxide (DMSO) (Sigma).

15. Hanks' Balanced Salt Medium (Life Technology).

2.3. Purification of Target Proteoglycans

1. Ni NTA agarose (Ni resin, Qiagen).

2. Equilibration buffer: 0.1 M NaCl, 44 mM NaHCO3, 1 mM imidazole, 0.5% Triton X-100 in H2O.

3. Met-free and Cys-free OPTI-MEM medium.

4. 10% Triton X-100 solution.

5. pH indicator paper.

6. Stringent wash buffer A:10 mM HEPES, 2 M NaCl, 10% glycerol, 0.1 mM PMSF, 2 mM imidazole, 0.5% Triton X-100, 6 M urea, 250 mM dithiothreitol in H2O, pH 8.0.

7. Stringent wash buffer B: 10 mM HEPES, 750 mM NaCl, 10% glycerol, 0.1 mM PMSF, 10 mM imidazole, 0.5% Triton X-100, 1 mg/mL bovine serum albumin in H2O, pH 8.0.

8. Stringent wash buffer C: 10 mM HEPES, 750 mM NaCl, 10% glycerol, 0.1 mM PMSF, 10 mM imidazole in H2O, pH 8.0.

9. Elution buffer: 10 mM HEPES, 750 mM NaCl,10% glycerol, 100 mM EDTA, 0.1 mM PMSF, 250 mM imidazole in H2O, pH 8.0.

10. Roto Torque heavy-duty rotator (Cole Parmer Instruments).

2.4. Identification of Target Proteoglycans

1. 5-15% linear gradient SDS-containing acrylamide gel (can be self-prepared or purchased from manufacturer).

2. Dithiothreitol (DTT), final concentration 38 mg/mL in loading buffer.

3. Gyrotory shaker (New Brunswick Scientific).

4. Gel fixing solution: 25% Methanol, 10% acetic acid in H2O.

5. Square Petri dish (100 x 100 x 15 mm, Nalge Nunc #4021).

6. Entensify Universal Autoradiography Enhancer Part A and Part B (Dupont).

7. Radiogram cassette and X-ray film (Kodak, Bio-Max films are best).

2.5. Characterization of Expressed Proteoglycans

1. Chondroitinase ABC (Seikagagu) (dissolved in buffer conditions recommended by the manufacturer).

2. 20x Chondroitinase ABC digest buffer: 2 M Tris-HCl, 600 mM sodium acetate, pH 8.0 (final conditions = 100 mM Tris-HCl, 30 mM sodium acetate, pH 8.0).

3. 10% Triton X-100 solution.

4. 100 mM N-ethylmaleimide (NEM) solution.

2.6. Detection of Proteoglycan Interaction with Heat-Shock Proteins Part I

1. Hsp25 antibody (Stressgen).

2. Protein A beads (Pierce).

3. Washing buffer D: 50 mM Tris-HCl, 1 M NaCl, 1% Triton X-100, pH 7.5.

2.7. Detection of Proteoglycan Interaction with Heat-Shock Proteins Part II

2. Washing buffer E: 130 mM NaCl, 20 mM Bicine, pH 8.0, ice cold.

3. Digitonin lysis buffer: 50 mM Bicine, pH 8.0, 150 mM NaCl, 5 mM KCl, 5 mM MgCl2, 0.2% digitonin, 1.5 mM DTSSP, ice cold.

4. 10 mM glycine solution, ice cold.

3. Methods

3.1. Construct Assembly of Target Proteoglycans

1. The target sequence was amplified by PCR using an upstream oligonucleotide (oligo 1) and a downstream oligonucleotide (oligo 2). Oligo 1 should contain a specific restriction site, which corresponds to one of the sites in the polylinker region in the eukaryotic expression vector. Oligo 2 should contain a specific restriction site, a His6 sequence (engineered for purification purposes), and a stop codon. The restriction site in oligo 2 can be the same as in oligo 1 or can be different, but it must correspond to a site in the polylinker region in the eukaryotic expression vector in correct orientation. Usually it is easier to have two different restriction sites so that you don't have to verify the orientation of the sequence after its insertion (see step 22).

2. The PCR reaction is usually a Hot-Start program (see Note 2). A portion of the PCR products (usually 5 ^L from a 50-^L PCR reaction) is denatured and electrophoresed in an ethidium bromide agarose gel to verify the size of the products.

3. Correct PCR products (the right size) are digested with Srfl and ligated to predigested pCR-Script SK(+) vector (Stratagene), which is then used to transform E. coli XL1-blue MRF' Kan supercompetent cells (following the manufacturer's manual).

4. Prepare LB broth following the manufacturer's instructions.

5. Dissolve X-gal in N,N-dimethyl formamide (20 mg/mL) and IPTG in water (50 mg/mL).

6. Place transformed products on an ampicillin-containing agar plate (containing X-gal and IPTG) and incubate at 37°C for 16 h. Blue and white colonies should appear on the plate.

7. Select white E. coli colonies, inoculate one colony into a 15-mL Falcon tube containing 3 mL of LB broth supplemented with ampicillin (50 ng/mL final concentration using 50 mg/mL 1000x stock), shake at 250 rpm at 37°C for 16 h (overnight).

8. Purify miniprep DNA using Wizard Plus Minipreps (Promega) following the manufacturer's instructions.

9. Digest purified DNA (5 ^L from 50 ^L) with appropriate restriction enzymes at 37°C for 1-2 h.

10. Electrophorese digested DNA in a 0.8-1% agarose gel to detect and select suitable DNA fragments, affirming the correct size of DNA fragments.

11. Digest the DNA fragments (16 ^L) again with the same restriction enzymes as in step 8.

12. Digest the pcDNA3 vector (Invitrogen) with the same restriction enzymes.

13. Electrophorese (10) and (11) in a 0.8-1% agarose gel.

14. Cut out the agarose bands containing the correct DNA fragments.

15. Purify the DNA using NanoSep filters following the manufacturer's instructions.

16. Ligate purified DNA fragments and pcDNA3 vector using Invitrogen T4 DNA ligase following the manufacturer's instructions.

17. Transform E. coliT0P10F' supercompetent cells using (16) following the manufacturer's instructions.

18. Repeat steps 7-10 to verify DNA inserts. Store bacteria-containing liquid broth in an 80/20 ratio glycerol at -80°C.

19. Strip bacteria onto an ampicillin-containing agar plate from glycerol stock and incubate overnight at 37°C for 16 h (overnight).

20. Inoculate one colony into a 125-mL glass flask containing 50 mL of LB broth supplemented with ampicillin (50 ng/mL final concentration) and shake at 250 rpm on a bacterial incubation shaker at 37°C for 16 h (overnight).

21. Prepare a maxiprep using QiaFilter DNA purification systems (Qiagen) following the manufacturer's instructions.

22. Quantify the DNA concentration and sequence the DNA to verify the correct sequence. The sequence must be 100% identical to the original design at the amino acid level.

23. The verified DNA is ready for the next step (transfection).

3.2. Expression of Target Proteoglycans

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