Prostar hplc system

Recombinant porcine amelogenins (rP172 and rP148) were expressed in Escherichia coli strain BL21-codon plus (DE3-RP, Agilent Technologies, Inc., Santa Clara, CA) as previously described.^{30 (link)–33 (link)} Protein purification was accomplished on a reverse phase C4 column (10 × 250 mm, 5 μm) mounted on a Varian Prostar HPLC system (ProStar/Dynamics6, version 6.41 Varian, Palo Alto, CA), using a linear gradient of 60% acetonitrile at a flow rate of 1.5 mL/min. Recombinant porcine amelogenin rP172 is an analogue of full-length native porcine P173, which has 173 amino acids, but rP172 lacks the N-terminal Met and a phosphate group on Ser16.^{34 (link)} The recombinant rP148 lacks the hydrophilic C-terminal. The C-terminal (M149-D173) 25 amino acid residue peptide was synthesized at the Microchemical Core Laboratory at the University of Southern California, using a Pioneer peptide synthesizer (Applied Biosystems, Foster City, CA) following the N-Fmoc-l-amino acid pentafluorophyenyl ester/HOBt coupling method.^{35 (link)}

The MatB-catalyzed synthesis of extender units has been previously described.^{37 (link), 38 (link), 44 (link)} Briefly, reactions were performed in a 50 μL reaction mixture containing 100 mM sodium phosphate (pH 7), MgCl₂ (2 mM), ATP (12 mM), coenzyme A (8 mM), malonate or corresponding analogue (16 mM) and wild-type or mutant MatB (10 μg) at 25 °C. Aliquots were removed after 3 h incubation, and quenched with an equal volume of ice-cold methanol, centrifuged at 10,000 g for 10 min, and cleared supernatants used for HPLC analysis on a Varian ProStar HPLC system. A series of linear gradients was developed from 0.1% TFA (A) in water to methanol (HPLC grade, B) using the following protocol: 0–32 min, 80% B; 32–35 min, 100% A. The flow rate was 1 mL/min, and the absorbance was monitored at 254 nm using Pursuit XRs C18 column (250 × 4.6 mm, Varian Inc.). To ensure complete conversion, the malonate analog and the acyl-CoA product HPLC peak areas were integrated, and the conversion (%) calculated as a percent of the total peak area. Product elution times and LC-MS data (data not shown) were in complete agreement with that previous described.^{37 (link), 44 (link)}