| The yield of recombinant proteins which incrementally used in industry and clinic at present,was severely inadequate.The establishment of a rapid and efficient purification technology system is essential to solve the problem.Recently,phase separation tags have been used for the purification of recombinant proteins,but each has its disadvantages.Calcium ion phase transition tags are used to purify individual proteins,but the investigation of removal of tags and the effect of cosolvent EDTA on protein structure and function are scarce.In this study,we tried to use human annexin A1 as an N-terminal fusion tag to investigate its separation and purification efficiency and the effect of EDTA on the activity of the separated fusion protein.Some reporter proteins,including three fluorescent proteins and two colored proteins with chromophore,were selected to visually study the separation effect.Using green fluorescent protein,a widely used protein,as the target protein,the optimal separation condition and the effect of combination and elution for different resins after separation were studied.The results showed that the purification effect of the green fluorescent protein fused with human annexin A1 was the highest.The additive cosolvent EDTA can chelate of the Fe-S cluster in Zea mays sirohydrochlorin ferrochelatase,m SF.Furthermore,four metal ion-dependent enzymes were selected for separation.The results showed that the effect of human annexin A1 tag on the separation of different enzymes was different,and the cosolvent EDTA inactivated the enzyme.Histidine tags coupled with human annexin A1 or C-terminal fusion affinity peptide tags facilitate further purification of the fused proteins separated proteins.This laid the foundation for designing different separation tags and purification tags for efficient purification of target proteins and prolonging the column efficiency.In this study,we established a rapid,efficient and inexpensive purification technology system using human annexin A1 as an N-terminal fusion tag. |
