| Purification and refolding. The previously described SDF-1a purification (13) has been modified to the following two column procedure. All purification steps were performed at 22C. Cells from a 10-L fermentation (165 g wet cell wt) were resuspended in 1 L of lysis buffer: 50 mM Tris –HCl (pH 8.0), 100 mM NaCl, 5 mM DTT, and 1 mM EDTA. The solution was mixed for 2 min with a polytron homogenizer (Brinkmann) and then disrupted with two passes through an APV Rannie mini-lab homogenizer at 10,000 psi. The disrupted cells were centrifuged for 25 min at 23,000g. The pellet was subjected to three wash steps (the first and third step with lysis buffer, the second step with lysis buffer 0.1% NP-40) followed by a final pellet solubilization in freshly prepared 8 M urea. Each 1-L wash step included a gentle polytron disruption of the pellet followed by a centrifugation step for 25 min at 23,000g. The final pellet was dissolved in 200 ml of a buffer, 50 mM Tris –HCl (pH 8.0), 8 M urea, 100 mM DTT, and 1 mM EDTA, and stirred overnight. The 8 M urea soluble fraction was complexed batchwise to 200 ml of SP Sepharose FF in 3.4 L of SP binding buffer: 25 mM Tris –HCl (pH 8.0), 8 M urea, and 20 mM 2- mercaptoethanol. The SP sepharose FF suspension was mixed overnight. The resin was separated from the unbound fraction by using a Buchner funnel. The SP Sepharose was washed with 800 ml of SP binding buffer and then filtered to semidryness. The resin was transferred into 8 L of refolding buffer: 50 mM Tris –HCl (pH 8.0), 100 mM NaCl, 0.1 mM reduced glutathione, and 0.1 mM oxidized glutathione. After 2 h, the resin was washed with 2 L of refolding buffer in the Buchner funnel. SDF-1a was eluted with 0.75 L of elution buffer: 25 mM Tris –HCl (pH 8.0) and 1.5 M NaCl. The SP Sepharose eluate was filtered through a 0.45-um cellulose acetate membrane and was adjusted to 0.1% TFA. Approximately 30 mg of the SP Sepharose eluate was loaded onto a reversed-phase Poros R2/H 16/100 column. The column was subjected to a 10 column vol linear gradient from 20 to 80% acetonitrile in 0.1% TFA. The HPLC step was repeated 6 times to process the entire sample. The fractions containing SDF-1a(95% purity) were lyophilized, resuspended in PBS buffer, and stored at -80C. SDS–PAGE, mass spectrometry, and protein sequencing all demonstrated that the protein purity was 95%. The amino acid sequence of the purified protein has been previously reported (13).
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