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Expression, purification, and characterization of a novel Ca(2+)- and phospholipid-binding protein annexin B2.

ABSTRACT: 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.

SUBMITTER: Lu YM

PROVIDER: S-EPMC2815297 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Expression, purification, and characterization of a novel Ca(2+)- and phospholipid-binding protein annexin B2.

Lu Yi-Ming YM Wang Na N Wang Jun-Jie JJ Wang Kai-Hui KH Sun Shu-Han SH

Molecular biology reports 20090520 3

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 prod ...[more]

PMID: 19455404

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Project description:AimSpike protein of coronavirus is responsible for virus binding, fusion and entry, and is a major inducer of neutralizing antibodies. This paper was to find a soluble and functional recombinant receptor-binding domain of severe acute respiratory syndrome-associated coronavirus (SARS-Cov), and to analyze its receptor binding ability.MethodsThree fusion tags (glutathione S-transferase, GST; thioredoxin, Trx; maltose-binding protein, MBP), which preferably contributes to increasing solubility and to facilitating the proper folding of heteroprotein, were used to acquire the soluble and functional expression of RBD protein in Escherichia coli (BL21(DE3) and Rosetta-gamiB(DE3) strains). The receptor binding ability of the purified soluble RBD protein was then detected by ELISA and flow cytometry assay.ResultsRBD of SARS-Cov spike protein was expressed as inclusion body when fused as TrxA tag form in both BL21 (DE3) and Rosetta-gamiB (DE3) under many different cultures and induction conditions. And there was no visible expression band on SDS-PAGE when RBD was expressed as MBP tagged form. Only GST tagged RBD was soluble expressed in BL21(DE3), and the protein was purified by AKTA Prime Chromatography system. The ELISA data showed that GST/RBD antigen had positive reaction with anti-RBD mouse monoclonal antibody 1A5. Further flow cytometry assay demonstrated the high efficiency of RBD's binding ability to ACE2 (angiotensin-converting enzyme 2) positive Vero E6 cell. And ACE2 was proved as a cellular receptor that meditated an initial-affinity interaction with SARS-Cov spike protein. The geometrical mean of GST and GST/RBD binding to Vero E6 cells were 77.08 and 352.73 respectively.ConclusionIn this paper, we get sufficient soluble N terminal GST tagged RBD protein expressed in E.coli BL21(DE3); data from ELISA and flow cytometry assay demonstrate that the recombinant protein is functional and binding to ACE2 positive Vero E6 cell efficiently. And the recombinant RBD derived from E.coli can be used to developing subunit vaccine to block S protein binding with receptor and to neutralizing SARS-Cov infection.

Dataset Information

Expression, purification, and characterization of a novel Ca(2+)- and phospholipid-binding protein annexin B2.

Publications

Expression, purification, and characterization of a novel Ca(2+)- and phospholipid-binding protein annexin B2.

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