Expression, purification, and characterization of a novel Ca(2+)- and phospholipid-binding protein annexin B2.
Annexin B2 (AnxB2) is a novel member of the annexin family of Ca(2+)- and phospholipid-binding proteins from Cysticercus cellulosae. To obtain highly pure AnxB2 with an easy and inexpensive purification approach, its cDNA was cloned into the prokaryotic expression vector pJLA503 and the translation initiation codon was immediately under the control of the inducible bacteriophage lambda promoters P(R) and P(L). After induction by shifting temperature, large amounts of non-fusion protein were produced in Escherichia coli in a soluble form. Then a novel purification method based on Ca(2+)-dependent phosphatidylserine (PS)-binding activity was established, whereby the purity of AnxB2 was increased to 98.7%. Western blot analysis showed that recombinant AnxB2 was specifically recognized by serum of pigs infected with cysticercosis. In vitro test showed that, the recombinant AnxB2 had anticoagulant activity and platelet binding activity. The expression, purification, and initial characterization of AnxB2 set an important stage for further characterization of the protein.
Lu
Yi-Ming
YM
Department of Medical Genetics, Second Military Medical University, Shanghai, China.
Wang
Na
N
Wang
Jun-Jie
JJ
Wang
Kai-Hui
KH
Sun
Shu-Han
SH
D000595:Amino Acid Sequence; D000818:Animals; D017302:Annexins / Q000235:genetics* / Q000378:metabolism; D010582:Bacteriophage lambda; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003552:Cysticercus / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005822:Genetic Vectors; D015801:Helminth Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D020539:Sequence Analysis, Protein
D017302:Annexins; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D015801:Helminth Proteins