The Expression In Escherichia Coli, Purification And Principal Characterization Of A New Six Histidines Tag Protein Annexin B1

Annexins are a large family of calcium-dependent phospholipid-binding proteins widely distributed in eukaryotes. Proteins of this family have been implicated in a wide range of important biological processes, such as anticoagulation, inhibition of phospholipase A2 and interaction with cytoskeletal proteins.Annexin B1, which is isolated from Cysticercus cellulosae, is a new member of the annexin family. It has the ability of anticoagulation and important therapeutic potential because of its thrombus targeting and antithrombotic properties. Furthermore, annexin B1 is one of a few annexins which was found in parasites. It is a protective antigen for vaccine development against cysticercosis.Purpose: In the previous researches, most of the attention concentrated on the expression and purification of GST-fusion and non-fusion annexin B1. However, both of them have some disadvantages when applied to protein crystallization. For example, the GST-tag is a protein as large as 23KD. Therefore, if it is not removed, the GST carrier may interfere with physiological function and activity of the interest protein. If the GST-tag is cleaved, a new protein contamination would be introduced. Therefore, it is not appropriate that taking the GST-fusion annexin B1 as the subject investigated. Although non-fusion annexin B1 also maintains the biological activity of wild-type protein, its major deficiency is that the N-terminal of protein is apt to degrade. In addition, the purification procedures of non-fusion protein are tedious. Thus, in order to get a group of new fusion-expressed annexin B1 with high stability, activity and purity, six histidines affinity tag (6×His tag) was added to the N-terminal of annexin B1.Method: In this experiment, it was chosen that adding six histidines affinity tag to the N-terminal of annexin B1. It is described a two-step chromatographic procedure for the purification of His-tag fusion annexin B1 by metal affinity chromatography as the capture step and size exclusion chromatography as the polishing step. Meanwhile, by KPTT clotting assays and the detection of apoptosis, it is certified 6×His fusion annexin B1 keeps calcium-dependent phospholipid-binding ability and maintains the same biological activities with wild-type annexin B1.Result: We finally got 23.0mg of pure annexin B1 which was bioactive just as it wild type's from 1000ml culture, and the homogeneity achieved 98.5%. It is certified 6×His fusion annexin B1 still keeps calcium-dependent phospholipid-binding ability and maintains the same biological activities with wild-type annexin B1.Conclusion: The experimental results show that a group of new fusion-expressed annexin B1 with high stability, activity and purity has been produced. Therefore, it has laid a solid foundation for the further study of protein crystallization.