Non-fusion expression in Escherichia coli, purification, and characterization of a novel Ca2+- and phospholipid-binding protein annexin B1.
Annexin B1 is a novel member of the annexin family of Ca2+- and phospholipid-binding proteins from Cysticercus cellulosae. To obtain high quality annexin B1 for biochemical and biophysical analyses, 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. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 25 mg/L bacterial culture. Western blot analysis showed that recombinant annexin B1 was specifically recognized by serum of pigs infected with cysticercosis. Secondary structure predictions from circular dichroism spectroscopy indicated that alpha-helix is the main secondary structure of the protein. In anticoagulant assays, the recombinant non-fusion protein exhibited dose-dependent effects in modified kaolin partial thromboplastin time (KPTT) prolongation and doubled the clotting time of control human plasma at 60 microg/ml. The expression, purification, and initial characterization of annexin B1 set an important stage for further characterization of the protein.
Zhang
Yi
Y
Department of Medical Genetics, The Second Military Medical University, Xiang Yin Road 800, Shanghai 200433, PR China.
Guo
Ying-Jun
YJ
Sun
Shu-han
SH
Yan
Hong-li
HL
He
Yan
Y
D000818:Animals; D017302:Annexins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D000925:Anticoagulants / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003552:Cysticercus / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics; D015801:Helminth Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D010314:Partial Thromboplastin Time; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology
D017302:Annexins; D000925:Anticoagulants; D018076:DNA, Complementary; D015801:Helminth Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C449006:annexin B1, Taenia solium; D005982:Glutathione Transferase