Extracellular production, purification and tag-removal of the N-terminally annexin- and ELP-tagged fluorescent proteins

Abstract

Background Rapid and cost-effective purification of the target protein for extracellular production in Escherichia coli is still challenge. Previously, we identified that human annexin A1 as a N-terminal fusion tag for Ca2+-dependent phase transition to simply, rapidly and cost-effectively purify three fluorescent proteins including emerald green fluorescent protein (EmGFP), red fluorescent protein mCherry, and flavin-binding cyan-green fluorescent protein.Results When the phage lytic protein was induced later, the annexin A1 tagged EmGFP was leaked into the culture, but purification efficiency was relatively low. Pre-overexpression of Bacillus cereus phospholipase C facilitated intracellular production of the fusion protein, and purified fusion protein showed the purity higher than other two fluorescent protein fusions. Using the co-expression system, the elastin-like polypeptide (ELP) tagged three fluorescent proteins via extracellular production were also purified in revisable protein precipitation. The yield of the purified annexin A1 tagged protein was comparable to that of the purified ELP tagged one. The silica-binding peptide tagged annexin A1-EmGFP bound to silica particles, and the ELP tagged mCherry strongly bound to phenyl sepharose was efficient for column-dependent purification. The extracellular nine tobacco etch virus protease variants with the annexin A1 tag were purified and the cleavage activity was assayed. Using the purified protease variant with the highest activity, the purification tag was removed in solution, or by on-resin cleavage of the immobilized annexin A1 or ELP tagged EmGFP. The soluble annexin A1-EmGFP with the Bacillus amyloliquefaciens alpha-amylase signal peptide was poorly produced in Bacillus subtilis, and the fusion protein with the α-factor signal peptide was located in intracellular Pichia pastoris.Conclusions The annexin A1 or ELP fusions in the culture were purified by revise transition cycles. On-resin cleavage facilitated removal of the reagents for protein purification, and fusion tag. However, the annexin A1-EmGFP fused the correspondent signal peptides displayed poor secretion efficiency in B. subtilis and P. pastoris. The platform will be used for simply and cost-effectively purifying the target proteins with industrial and clinical values without cell disruption process, and rapidly testifying the activity of the engineered enzyme variants.