Maltose-binding Protein Improving the Crystallizability of C2 Domain of Human Coagulation Factor V

Human coagulation Factor V（FV）, together with Factor Xa, assembles to prothrombinase complex on activated cell surface, which converts prothrombin into thrombin, leading to fibrin deposition. The C2 domain of FV is believed to be a primary anchor for the assembly of pro- thrombinase on the cell surface, and was proposed as a target to intervene with pathological thrombotic events. We report here the crystal structure of the C2 domain of FV fused to maltose-binding protein（MBP）. The fusion tag of MBP is critical to generate the crystal for this study. There is no strong interaction between MBP and FVC2. The overall structure of FVC2 is similar to the previous FVC2 structures, suggesting the MBP fusion does not perturb the molecular structure of FVC2. This crystal form of FVC2 can be used for future study of molecular interaction between FVC2 and its inhibitors.

Immunization of Male Mice with a New Recombinant GnRH Fusion Protein Reduces the Testicular Function

The objective of the present study was to evaluate the effect of an immunogenic maltose-binding protein-gonadotropin releasing hormone （GnRH-6-MBP） using genetic engineering. The synthetic mammalian tandem repeated GnRH hexamer gene was inserted into the expression plasmid pMAL-c2x. Recombinant GnRH-6-MBP protein was over- expressed in E.coli strain BL21. Amylose resin with affinity chromatograph was used to purify target protein. The reactiongenicity of fusion protein was identified by indirect enzyme linked immunosorbent assay （ELISA）, and the antigenicity and biological effects of GnRH-6-MBP were tested in mice. In the experiment, 20 male Kunming white mice of 20 d old were randomly divided into treatment and control group. Ten mice were immunized with 100 μg GnRH-6-MBP administered subcutaneously （s.c.） thrice at 2-week intervals with GnRH-6-MBP. Mice were sacrificed after 3 weeks following the booster injection, the testis was removed, weighed and measured, and the histological structure was observed. The reactiongenicity of fusion protein to GnRH antibody was much higher than the control. Active immunization against GnRH-6-MBP reduced remarkably （P 〈 0.01） the length and weight of the testis, and shortened the girth and width of the testis （P 〈 0.05）, and suppressed testicular spermatogenesis compared to the control mice. These results indicate that the recombinant GnRH-6-MBP acted as a strong immunogen and caused atrophy of the testis.

Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli

AIM: To produce a recombinant protein rMBP-NAP, which was fusionally expressed by Helicobacter pylori(H pylori)neutrophil-activating protein (NAP) and E. coli maltosebinding protein (MBP) and to evaluate its immunoreactivity and immunogenicity.METHODS: Neutrophil-activating protein gene of H pylori (HP-napA) was subcloned from the recombinant plasmid pNEB-napA, and fused to MalE gene of expressing vector pMAL-c2x. The recombinant plasmid pMAL-c2x-napA was confirmed by restriction enzyme digestion, and then transformed into E. coli TB1. Fusion protein rMBP-NAP was induced by IPTG and identified by SDS-PAGE analysis.Soluble rMBP-NAP was purified by amylose affinity chromatography. Immunoreactivity and immunogenicity of the fusion protein were evaluated by animal experiment,Western blotting with human H pylori anti-sera.RESULTS: E.coli TB1 carrying recombinant plasmid pMAL-c2x-napA was constructed and led to a high efficiency cytosol expression of fusion protein rBMP -NAP when induced by IPTG.The molecular weight of rBMP-NAP was about 57 kD,accounting for 37.55% of the total protein in the sonicated supematant of E. coli TB1 (pMAL-c2x-napA). The purity of the fusion protein after one-step affinity chromatography was 94% and the yield was 100 mg per liter of bacterial culture.The purified fusion protein could be specifically recognized by both human anti-sera from clinical patients with H pylori infection and rabbit sera immunized by rMBP-NAP itself.CONCLUSION: Recombinant protein rMBP-NAP might be a novel antigen for vaccine development against H pylori.

Expression,purification and identification of LBD domain of human PPARδ in E.coli

Peroxisome proliferator-activated receptors （PPARs） are ligand-activated transcription factors known to play a pivotal role in regulations of metabolism. In order to yield soluble ligand binding domain of PPARδ （PPARδLBD） for screening ligands, the cDNA was amplified using total RNA from HepG2 cells by RT-PCR. Then the enzyme-digested product was inserted . downstream of the malE gene in the vectorpMAL-p2x, which encoded maltose-binding protein （MBP）, resulting in the expression of an MBP-PPARδLBD fusion protein. The recombinant plasmid was transformed into E. coli TBI that was cultured shakily at 30 ℃, 200 r/min and induced by 0.4 mmol/L IPTG for 6 h. The cells were harvested by centrifugation and broken by sonication. The expressed fusion protein was soluble and accounted for 0.31 of the total protein in the supernatant. Western blot analysis showed that the expressed MBP-PPARδLBD could bind to anti-MBP-antibody. The MBP-PPARδLBD fusion protein of 77 kDa and the PPARδLBD protein of 34 kDa were obtained by amylose-resin affinity chromatography without or with digestion of Factor Xa. They were both homogeneity, judged by SDS-PAGE. The recombinant MBP-PPARδLBD and PPARδLBD protein with high purity is obtained, which provides the necessary material for screening and researching PPARδ ligands.

Purification of the Drosophila melanogaster Proteins Inscuteable and Staufen Expressed in Escherichia coli

The proteins Inscuteable and Staufen are key components during asymmetric cell division of neuroblasts for the development of Drosophila melanogaster. Expression and purification of both proteins has been a difficult task for structure-function studies. Based on codon optimization for protein expression in Escherichia coli, we have been able to produce, in soluble form, the C-terminal domains of Inscuteable and Staufen as chimeras with N-terminal maltose binding protein tag that contains a rigid linker between them for feasible crystallization. In addition, using an optimized synthetic gene, corresponding to the amino acid region 250 - 623 of Inscuteable fused to glutathione-S-transferase, low-scale expression experiments showed production of soluble protein. Finally, eukaryotic expression of Inscuteable in the methylothropic yeast Pichia pastoris failed to produce the Drosophila protein at detectable amounts, reinforcing the fact that E. coli still was the microorganism of choice for high-yield protein expression.