Improved extracellular expression and purification of recombinant Staphylococcus aureus protein A

• Secretory overexpression of Protein A in Escherichia coli was improved with help of additives.
• A fermentation protocol was developed which is suitable for the high yield production of SpA.
• The protein was purified by tangential flow filtration and ion exchange chromatography.
• IgG binding activity was retained during the process.

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 380 mg L−1 of protein A were obtained when Tris–HCl pH 8.5 was added up to a final concentration of 180 mM in shaking flask experiments. Based on these observations, a protocol was developed for the extracellular production of SpA in a stirred tank bioreactor yielding 5.5 g L−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.