Investigating the applicability of human annexin A1 as an affinity tag for separation and purification of the target proteins

Abstract

Backgrounds Rapid separation methods with fusion of the affinity tags have been developed. However, the affinity tag for simply and cheaply separating the fused target protein is still lacking. Results Separation conditions for the human annexin A1 (hanA1) tagged emerald green fluorescent protein (EmGFP) in Escherichia coli cytoplasm were optimized via precipitation with CaCl2 and re-solubilization with EDTA-Na2. Combination of the affinity precipitation with each of three affinity purification approaches increased the hanA1-EmGFP purity, and on-resin cleavage facilitated release of tag-free EmGFP. With addition of Triton X-100 to the culture, the fused EmGFP, red fluorescent protein mCherry, or the FMN-dependent fluorescent protein LOV, was also prepared with increasing CaCl2concentrations after it was secreted to the culture from E. coli, whereas the hanA1-EmGFP fused to the amyQ signal sequence was insolubly produced in Bacillus subtilis cells, and the hanA1-EmGFP fused to the α signal peptide produced in Pichia pastoris was unable to be secreted to the culture. The affinity separation was visualized by use of three fluorescent proteins including the EmGFP, mCherry and LOV, and two colored proteins including a bacterial hemoglobin, and maize sirohydrochlorin ferrochelatase (mSF) showing brown containing the [2Fe–2S] cluster. The added EDTA-Na2disrupted the mSF structure, and inhibited activities of the selected four metal-dependent enzymes, but showed little impact on two specific proteases for cleaving the fusion proteins. After affinity precipitation, the tagged lysine decarboxylase was prepared as cross-linked enzyme aggregates. Conclusion The hanA1 tag is ideal for simple, rapid and cost-effective separation of the target proteins via intracellular and extracellular production in E. coli. This tag is also used for further affinity purification of the selected proteins and enzymes potentially applied in industry and diagnosis.