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«1. Introduction The functions of many proteins are likely to be regulated by phosphorylation. Thus, antibodies that can recognize specifically ...»

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2

Generation and Application of Phospho-specific

Antibodies for p53 and pRB

Yoichi Taya, Kiichiro Nakajima, Kumiko Yoshizawa-Kumagaye,

and Katsuyuki Tamai

1. Introduction

The functions of many proteins are likely to be regulated by phosphorylation. Thus,

antibodies that can recognize specifically phosphorylated sites on proteins have a wide

variety of uses for studying the function and regulation of phosphoproteins. We have

improved methods for generation of phosphorylation site-specific antibodies and have successfully obtained antibodies for the analysis of most of the phosphorylation sites on p53 and RB proteins.

The RB protein (pRB) was first shown by Taya and colleagues to be phosphorylated by a cyclin-dependent kinase (Cdk) at multiple sites in vitro (reviewed in ref. 1). Subsequently a variety of novel Cdks and their inhibitory proteins have been identified, and it is commonly understood now that phosphorylation of pRB by Cdks plays a key role in the regulation of cellular proliferation and in cancer (1–3). In an attempt to elucidate the physiologic relevance of phosphorylation of pRB, it was decided to generate antibodies to recognize specific phosphorylation sites of pRB using chemically synthesized phosphopeptides as antigens. However, it was not easy to synthesize peptides containing stably phosphorylated serine or threonine. Therefore, we have improved methods for obtaining such peptides.

Taking advantage of this improved methodology, the production of antibodies to specific phosphorylation sites of pRB was initiated. After demonstrating that this approach is successful for pRB, we also embarked upon generation of a series of antibodies specific for specific phosphorylation sites of p53.

We generally synthesize phosphopeptides as shown in Fig. 1 for immunization of rabbits or mice. Cys is coupled to the N-terminus of most peptides to allow conjugation with KLH for more effective immunization. Because epitopes of antibodies can comprise as few as three or four amino acid residues, it is recommended that, when possible, only three residues be placed C-terminal to phosphoserine/threonine. In our experience we have observed that if there are more residues on this external side of the phosphorylated residue, antibodies directed against unphosphorylated epitopes are prefFrom: Methods in Molecular Biology, Vol. 223: Tumor Suppressor Genes: Regulation, Function, and Medicinal Applications. Edited by: Wafik S. El-Deiry © Humana Press Inc., Totowa, NJ 18 Taya, Nakajima, and Tamai Fig. 1. Schematic presentation of a phosphopeptide antigen.

erentially produced. It is acceptable to include 6 amino acids plus N-terminal Cys on the other (internal) side of the phosphorylated residue.

If the peptide is too hydrophobic this will render it insoluble, so it is better to replace 2 or 3 amino acids on the internal side (Cys side) with 2 or 3 Arg residues. For the purpose of chemical synthesis, internal Met or Cys residues should be avoided. When an inappropriate amino acid residue is located on the N-terminal side, it is recommended to exchange the length of the N-terminal side with that of the C-terminal side, putting the Cys residue for conjugation with KLH at the C-terminus.

1.1. Chemical Synthesis of Phosphopeptides Establishing methods for generating the most effective phosphopeptides is very important. To synthesize phosphopeptides, there are two basic strategies: (a) The prephosphorylation method, in which the protected phosphorylated amino acid derivatives are used as the building blocks of peptides; or (2) the postphosphorylation method, in which the unprotected hydroxy groups are phosphorylated on the completely assembled peptide chain. In this chapter we focus on the method we have used most extensively, which is the prephosphorylration procedure for the synthesis of desired phosphorylated peptides involving the t-butoxycarbonyl (Boc), and the 9-fluorenylmethoxycarbonyl (Fmoc) strategies.

2. Materials

2.1. Recommended Standard Synthesis of Phosphopeptides by the Boc Strategy (4,5)

1. Automated peptide synthesizer: ABI-430A (Applied Biosystems, Foster City, CA, USA).

2. Resin: Boc-amino acid preloaded PAM resin (Applied Biosystems, Foster City, CA, USA)

3. All of the Boc-amino acid derivatives Arg (Mts), Glu (OBzl), Asp (OBzl), Cys (MeOBzl), His (Bom), Lys (Cl-Z), Ser (Bzl), Thr (Bzl), Tyr (Br-Z), Trp (Hoc) were purchased from Peptide Institute (Osaka, Japan), and the phosphorylated, Boc-Ser (ALL2) and Boc-Thr (PO3All2) were synthesized for our own uses (6,7).

Phospho-specific Antibodies for p53 and pRB 19

4. Preparative reversed-phase (RP)-HPLC: YMC-Pack ODS-AM (30 250 mm, YMC, Kyoto, Japan) using Shimdzu LC-8 A HPLC Apparatus.

2.2. Recommended Standard Synthesis of Phosphopeptides by the Fmoc Strategy (8)

1. Automated peptide synthesizer: ABI-433A (Applied Biosystems, Foster City, CA, USA).

2. Resin: Fmoc-amino acid preloaded p-hydroxymethyl-phenoxymethylated resin (Wang resin) (0.25-mmol sacle) (Applied Biosystems, Foster City, CA, USA).

3. All of the Fmoc-amino acid derivatives were purchased from Peptide Institute (Osaka, Japan).

4. Arg (Pbf), Glu (OtBu), Asp (OtBu), Cys (Trt), His (Trt), Lys (Boc), Ser (tBu), Thr (tBu), Tyr (tBu), Trp (Boc), and the phosphorylated Fmoc-amino acids, Fmoc-Ser(PO(OH)OBzl) and Thr(PO(OH)OBzl), were purchased from Watanabe Chemical Industries (Hiroshima, Japan) or Novabiochem (Laufelfingen, Switzerland).





5. Preparative RP-HPLC: YMC-Pack ODS-AM (30 250 mm, YMC, Kyoto, Japan) using Shimdzu LC-8 A HPLC apparatus.

3. Methods

3.1. Recommended Standard Synthesis of Phosphopeptides by the Boc Strategy (4,5) 3.1.1. Synthesis of Protected Peptide Resin by the Boc Strategy The elongation of the desired protected peptide resin was carried out by the 0.5-mmol scale standard protocol of the benzotriazol active ester method in the system software version 1.40. The synthesis starts with the first Boc-amino acid attached to the PAM resin.

After deprotection (50% trifluorocetic acid [TFA]/dichloromethane [DCM] (v/v) of the Boc group of first amino function, the next Boc-amino acid was coupled (N,N -dicyclohexylcarbodiimide [DCC]/N-hydroxybenzotriazole [HOBt]). Consecutive deprotection/coupling steps accomplished chain elongation. The finally obtained protected peptide resin was dried and used for the following deprotection procedure (see Note 1).

3.1.2. Deprotection and Cleavage Procedure by TFMSA To the peptide resin (0.3-mmol scale) were added thioanisole (36 mmol, 120 eq), pcresol (36 mmol, 120 eq), trifluoromethanesulfonic acid (TFMSA) (36 mmol, 120 eq), and TFA (30 mL) in a round-bottom flask (500 mL) with stirring at 2 0 (see Note 2).

After the reaction mixture was stirring for 2.5 h at 2 0, the reaction mixture was diluted by dry ether (300 mL) at 2 0. Thus obtained precipitate was filtered off by the aid of a microfilter, and the crude phosphopeptide was extracted by TFA to separate the resin. Dry ether was added again to the TFA solution obtained, and the crude phosphopeptide precipitate was filtered off and dried over NaOH in vacuo.

3.1.3. Purification Procedure To a solution of thus obtained crude compound (0.3 mmol) in water (20 mL) was added dithiothreitol (DTT) (1.5 mmol), and the pH of the solution was adjusted to pH 8–9 with dilute ammonia aqueous to prepare the thiol-free peptide (see Note 3). After the mixture was allowed to stand at room temperature for 10–20 min, the reduction reacTaya, Nakajima, and Tamai tion was stopped by addition of TFA. The mixture was immediately purified by preparative RP-HPLC (CH3CN/0.1% TFA aq. linear gradient system). The desired fractions were combined and lyophylized to obtain the purified titred phosphopeptide.

Isolated peptide was identified by amino acid analysis and mass spectrometry. The purity of the final product was inspected by analytical RP-HPLC.

3.2. Recommended Standard Synthesis of Phosphopeptides by the Fmoc Strategy (8) 3.2.1. Synthesis of the Protected Peptide Resin by the Fmoc Strategy The elongation of the desired protected peptide resin was carried out by the 0.25mmol-scale standard protocol of the 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/diisopropylethylamine (DIPEA) method in the system software. The synthesis starts with the first Fmoc-amino acid atached to the Wang resin (0.25 mmol).

After deprotection (piperidine/dimethyl sulfoxide (DMF), (1/4 v/v) of the Fmoc group of the first amino function, the next Fmoc amino acid was coupled (HBTU/ DIPEA). Consecutive deprotection/coupling steps accomplished chain elongation. The finally obtained protected peptide resin was dried and used for the following deprotection procedure.

3.2.2. Deprotection and Cleavage Procedure by TFA To the peptide resin (0.25 mmol) were added TFA (20 mL), triisopropylsilane (TIS) (0.54 mL), H2O (0.54 mL), and thiophenol (0.54 mL) (92.5/2.5/2.5/2.5, v/v) in a roundbottom flask (500 mL) with stirring at room temperature.

After the reaction mixture was stirring for 1.5–2 h at room temperature, the reaction mixture was diluted by dry ether (300 mL). The precipitate obtained was filtered off with a microfilter, and the crude phosphopeptide was extracted with 0.1% TFA to separate the resin. The extracted solution was lyophylized to obtain the desired crude phosphopetide.

The crude product was purified in the same way as described for the Boc Strategy.

3.3. Generation of Phospho-specific Antibodies 3.3.1. Coupling Peptide to Carrier Protein with MBS

1. Dissolve keyhole limpet hemocyanin (KLH) to a concentration of 16 mg/mL in 1 mL of 10 mM sodium phosphate buffer, pH 7.2 (see Note 4).

2. Prepare 280 mg/mL of m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) in dimethylformamide freshly.

3. Add 10 lL of MBS with stirring to avoid a high local concentration and continue to stir for 30 min at room temperature.

4. Centriguge at 15,000 rpm at 4 C for 5 min. Take a supernatant.

5. Separate the MBS-activated KLH from the free MBS by gel filtration on Sephadex G25 equilibrated with 50 mM sodium phosphate buffer, pH 6.0.

6. Pool MBS-activated KLH and divide equally into 10 microcentrifuge tubes. Store at 80 C until use.

7. Thaw MBS-activated KLH in one tube.

8. Add 0.5 vol of 0.2 M Na2HPO4 (pH should be 7.3–7.5).

Phospho-specific Antibodies for p53 and pRB 21

9. Dissolve 1 mg of the peptide containing cysteine residue at the N- or C-terminus in 0.5 mL of distilled water, and add to MBS-activated KLH.

10. Stir the reaction for 3 h at room temperature.

11. Divide equally into 10 microcentrifuge tubes. Store at 80 C until use.

3.3.1.1. DEOXIDATION PEPTIDE OF

When free sulfhydryl residue in the peptide seems to be oxidized, the peptide should be deoxidized by the following protocol. (Ellman’s reagent can be used to determine if there are free sulfhydryls available on the terminus of the peptide.)

1. Dissolve 1 mg of the peptide containing cysteine residue at the N- or C-terminus in 0.5 mL of 20 mM sodium phosphate buffer, pH 8.5.

2. Add NaBH4 to a final concentration of 0.1 M.

3. Stand the reaction for 45 min at room temperature.

4. Adjust the final pH to 4.0 by adding 0.1 N HCl.

5. Stand the reaction for 5 min at room temperature.

6. Adjust the final pH to 7.5 by adding 0.2 M Na2HPO4.

7. Add to MBS-activated KLH.

8. Stir the reaction for 3 h at room temperature.

9. Dialyze against phosphate-buffered saline (PBS) at 4 C overnight.

10. Divide equally into 10 microcentrifuge tubes. Store at 80 C until use.

3.3.2. Production of Anti-phosphopeptide Polyclonal Antibody 3.3.2.1. IMMUNIZATION RABBITS OF Two or more female rabbits (weight ~2.5 kg) were immunized subcutaneously by standard protocol with ~100 lg of phosphopeptide-KLH conjugate emulsified with Freund’s complete adjuvant (FCA) 4 times at biweekly intervals, and boosted with phospho-peptide-KLH conjugate in Freund’s incomplete adjuvant (see Note 5). One week after boost, bleed approx. 70 mL from marginal ear vein of rabbits.

3.3.2.2. AMMONIUM SULFATE PRECIPITATION

1. Incubate rabbit antiserum (~30 mL) for 30 min at 56 C (heat inactivation).

2. Centrifuge at 15,000 rpm at 4 C for 5 min.

3. Transfer the supernatant to an appropriate vessel.

4. Determine the volume of serum and add an equal volume of PBS.

5. Add slowly 0.8 vol of saturated ammonium sulfate solution.

6. Stir gently for 15 min at room temperature.

7. Centrifuge at 15,000 rpm at 4 C for 15 min.

8. Discard the supernatant.

9. Resuspend the precipitate with PBS (2 vol of initial antiserum).

10. Repeat steps 4–7.

11. Resuspend the precipitate with PBS (0.5 vol of initial antiserum)

12. Dialyze against 50 vol of PBS using dialyzing tubing at 4 C overnight (3 changes).

13. Centrifuge at 15,000 rpm at 4 C for 15 min for remove any remaining debris.

3.3.2.3. COUPLING PEPTIDE-CONTAINING CYSTEINE RESIDUE GEL

THE TO THE

1. Pack 1 mL of iodeacetyl immobilized gel (SulfoLink coupling gel: Pierce, Rockford, IL, USA) into appropriate-sized column (see Note 6).

2. Equilibrate the column with 6 column vols of 50 mM Tris-HCl, pH 8.5, and 5 mM EDTA-Na.

22 Taya, Nakajima, and Tamai

3. Dissolve 2 mg of the peptide containing cysteine residue at the N- or C-terminus in 1 mL of 50 mM Tris-HCl, pH 8.5, and 5 mM EDTA-Na.

4. Apply the peptide solution on the column and rotate the column for 1 h at room temperature or overnight at 40 C.

5. Wash the column with 3 column vols of 50 mM Tris-HCl, pH 8.5, and 5 mM EDTA-Na.

6. Add 1 mL of 50 mM cysteine solution to the column to block any remaining active iodeacetyl groups, and rotate the column for 1 h at room temperature.

7. Wash the column with at least 15 column vols of 1 M NaCl and 15 column vols of 0.05% NaN3.

8. Equilibrate the column with 6 column vols of PBS containing 0.3 M NaCl.

3.3.2.4. AFFINITY CHROMATOGRAPHY

1. Equilibrate the phosphopeptide coupled gel in a column with 5 gel vols of PBS containing

0.3 M NaCl.



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