Detection of human annexin A1 as the novel N-terminal tag for separation and purification handle

Abstract

Background Several fusion tags for separation handle have been developed, but the fused tag for simply and cheaply separating the target protein is still lacking. Results Separation conditions for the human annexin A1 (hanA1) tagged emerald green fluorescent protein (EmGFP) in Escherichia coli were optimized via precipitation with calcium chloride (CaCl2) and resolubilization with ethylenediamine tetraacetic acid disodium salt (EDTA-Na2). The HanA1-EmGFP absorbing with other three affinity matrix was detected, only it was strongly bound to heparin Sepharose. The separation efficiency of the HanA1-EmGFP was comparable with purification efficiency of the His6-tagged HanA1-EmGFP via metal ion affinity chromatography. Three fluorescent proteins for the EmGFP, mCherry red fluorescent protein and flavin-binding cyan-green fluorescent protein LOV from Chlamydomonas reinhardtii were used for naked-eye detection of the separation and purification processes, and two colored proteins including a red protein for a Vitreoscilla hemoglobin (Vhb), and a brown protein for maize sirohydrochlorin ferrochelatase (mSF) were used for visualizing the separation process. The added EDTA-Na2 disrupted the Fe–S cluster in the mSF, but it showed little impact on heme in Vhb. Conclusions The selected five colored proteins were efficient for detecting the applicability of the highly selective hanA1 for fusion separation and purification handle. The fused hanA1 tag will be potentially used for simple and cheap affinity separation of the target proteins in industry and diagnosis.

Backbone Cyclization of Flavin Mononucleotide-Based Fluorescent Protein Increases Fluorescence and Stability

“…The pGST-tevS-EmGFP plasmid was constructed for expressing the fusion protein constituted of the His6-tagged glutathione S-transferase (GST), the incorporated tobacco etch virus protease (TEVp) cleavage sequence (tevS, ENLYFQ ↓ G), and green fluorescent protein (GFP) variant emerald GFP (EmGFP). The p28GFP plasmid with sequence coding for the His6-tagged EmGFP inserted into the pET-28b plasmid, the pGST-rpS-eDAL plasmid to express the fusion protein containing the GST tag, human rhinovirus 3C protease (R3P) cleavable sequence (rpS, LEVLFQGP), and the target protein eDAL, the pNpu vector for expressing the split intein variant, the phanA1-CrLOV plasmid for expressing the tagged codon-optimized CrLOV variant, and help vector pBAD33-hPDI were constructed previously [ 12 , 20 - 22 ]. The plasmid for expressing the His6-GST tagged R3P was gifted from Professor Jiangye Zang of the University of Science and Technology of China.…”

“…Protein concentration was analyzed by using the Bradford method, with bovine serum albumin as the standard. The CrLOV fluorescence on the SDS-PAGE gel was recorded [ 12 ]. Briefly, protein samples were mixed with the loading buffer and incubated at 37°C for 10 min.…”

“…CrLOV is a photostable, thermostable, and fast-maturing monomeric protein superior to other homologues in brightness and operational pH range [ 4 ]. Using the codon-optimized CrLOV reporter, we detected human annexin A1 as the purification tag in E. coli for Ca 2+ -dependent phase transition separation [ 12 ]. Like that from GFP and red fluorescent protein resistant to sodium dodecyl sulfate (SDS) at low concentration [ 13 ], a fluorescent band from CrLOV was observed under UV irradiation after SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was finished [ 12 ].…”

“…Using the codon-optimized CrLOV reporter, we detected human annexin A1 as the purification tag in E. coli for Ca 2+ -dependent phase transition separation [ 12 ]. Like that from GFP and red fluorescent protein resistant to sodium dodecyl sulfate (SDS) at low concentration [ 13 ], a fluorescent band from CrLOV was observed under UV irradiation after SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was finished [ 12 ]. To enhance the CrLOV fluorescence, other strategies are needed.…”