Production of biologically active recombinant annexin B1 with enhanced stability via a tagging system.
Annexin B1 is a novel Ca(2+)-dependent phospholipid-binding protein from metacestodes of Taenia solium and has been shown to have many potential biomedical applications. Although annexin B1 has been produced successfully in Escherichia coli, the purified protein has poor stability at room temperature, which has hindered our attempts to further study its structure-function relationship. To increase the stability of the protein, the construction and purification procedures were examined and changed to hopefully increase its effectiveness. In this study, we describe a new recombinant annexin B1 expressed with a hexahistidine tag fused to its N-terminal end, which was purified to homogeneity in two steps using immobilized metal affinity followed by size exclusion chromatography. The final yield was approximately 23 mg/L of bacterial culture. Isoelectric focusing and mass spectrometry analysis showed that the protein purified by this method was quite stable at room temperature, even greater than 3 days later. A series of functional tests indicated that the recombinant protein had high anticoagulant activity, and fluorescence-labeled annexin B1 could bind to the outer membranes of apoptotic mammalian cells and efficiently detect them in the early stages of apoptosis.
Zhang
Yi
Y
Department of Medical Genetics, The Second Military Medical University, Xiang Yin Road 800, Shanghai, China.
Zheng
Tao
T
Wang
Yue
Y
Guo
Yingjun
Y
Ding
Feixiang
F
Fei
Mingyu
M
Cui
Huixian
H
Sun
Shuhan
S
D000818:Animals; D017302:Annexins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017209:Apoptosis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D015801:Helminth Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007526:Isoelectric Point; D013058:Mass Spectrometry; D011485:Protein Binding; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D041201:Taenia solium / Q000235:genetics; D013997:Time Factors
D017302:Annexins; D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; C449006:annexin B1, Taenia solium