Non-fusion expression in Escherichia coli: Single-step purification of recombinant human annexin A5 for detection of apoptosis.
Recombinant human annexin A5 (rh-annexin A5) was originally used to detect early stages of apoptosis in vitro. With the development of radioactive labeling and imaging techniques, annexin A5 labeled with radioactive markers can play a more important role in monitoring apoptotic cells in vivo. To obtain highly pure rh-annexin A5 with an easy and inexpensive purification approach, we constructed a pJLA503-annexin A5 expression plasmid, which could overexpress human annexin A5 in a soluble form in Escherichia coli. Then a novel purification method based on Ca2+-dependent phosphatidylserine (PS)-binding activity was established, whereby the purity of rh-annexin A5 was increased to 98%. To confirm the PS affinity of rh-annexin A5 produced by this purification protocol, a simple and reliable lipid membrane model was prepared and used in the binding test. As a probe to detect apoptosis, the fluorescein isothiocyanate-labeled rh-annexin A5 was incubated with apoptotic cells. The results showed that the labeled rh-annexin A5 possessed high affinity for PS molecule and was able to indicate different apoptotic states.
Wang
Fang
F
Department of Medical Genetics, Second Military Medical University, No. 800 Xiang Yin Road, Shanghai 200433, China.
He
Xiao-Wen
XW
Yan
Hong-Li
HL
Huang
Jing-Jing
JJ
Zhang
Yi
Y
Jiang
Lei
L
Gao
Yuan-Jian
YJ
Sun
Shu-Han
SH
D017304:Annexin A5 / Q000235:genetics / Q000302:isolation & purification; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000737:chemistry; D002460:Cell Line; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D066298:In Vitro Techniques; D010743:Phospholipids / Q000737:chemistry / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D017304:Annexin A5; D010743:Phospholipids; D011994:Recombinant Proteins; D002118:Calcium