Protein A from Staphylococcus aureus plays one key role as an immobilized affinity ligand for the purification of antibodies. A simple method for its extracellular expression in Escherichia coli and subsequent purification is reported herein. The N-terminus of the gene coding for the five IgG binding domains was fused to a pelB signal peptide which is responsible for periplasmic localization and which is removed after translocation into the periplasmic space of E.coli. Different additives, which were added at the same time with the induction of the protein expression by IPTG, were tested in order to facilitate the release of the target protein. With help of this optimized release protocol, more than 380mgL(-1) of protein A were obtained when Tris-HCl pH 8.5 was added up to a final concentration of 180mM in shaking flask experiments. Based on these observations, a protocol was developed for the extracellular production of SpA in a stirred tank bioreactor yielding 5.5gL(-1) of the secreted target protein. After cell removal by centrifugation, the protein A-containing supernatant was concentrated and dialyzed by tangential flow filtration. The target protein was subsequently purified by anion exchange chromatography with a total process yield of 90% and a final purity of ⩾95% (RP HPLC) was achieved.
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*Note: The HRP-labeled Chicken Anti-Protein A used in this publication was produced by Gallus Immunotech Inc.
Chemical permeabilization, Extracellular expression, Recombinant protein A, pelB