BIN1b

We have previously shown that the small metal-binding proteins CusF3H+ and SmbP can be used as fusion proteins for the expression and purification of recombinant proteins in Escherichia coli. Because of their small size, both around 10 kDa, they are suitable for the production of peptides to avoid meager yields after the final purification step of tag removal. Bin1b is a beta-defensin found in the epididymis of rats that has shown to have antimicrobial activity. Previous methodologies used to express this antimicrobial peptide in E. coli involve the expression of the peptide as inclusion bodies followed by in vitro refolding or the supplementation of the proteins necessary for proper folding of the peptide in the cytoplasm via a second plasmid. Here, we developed a methodology that forgoes these approaches and instead uses the fusion proteins CusF3H+ or SmbP and the E. coli strain SHuffle to obtain a soluble recombinant protein that contains the mature Bin1b peptide. The recombinant protein is purified using IMAC chromatography and is subsequently cleaved with enterokinase to separate the fusion protein from Bin1b. The purified peptide displays antimicrobial activity against E. coli, as previously shown. Furthermore, we also tested its antimicrobial activity against the Gram-positive bacteria Staphylococcus aureus and found that Bin1b is also capable of inhibiting the growth of this bacterium. In conclusion, we developed a practical methodology for the expression and purification of the bioactive Bin1b peptide in E. coli using the fusion proteins CusF3H+ and SmbP. This approach could be further applied for the production of more biologically active peptides.

Objectives: To study the anti-fertility effect of a DNA vaccine using Bin1b as the target antigen in male mice. Methods: A novel recombinant eukaryotic vector containing a fusion gene sequence of mouse Bin1b in tandem with three copies of C3d fragment (C3d3) was used to construct pSG.SS.C3d3.YL.Bin1b. The correct expression of the Bin1b-C3d3 protein was confirmed in transfected HEK293 cells by indirect immunofluorescence and western blot analysis. The fertility of immunised mice was determined by a mating experiment and sperm motility test. Anti-Bin1b antibody titres in sera were examined by ELISA assays. Binding activity of C3d3 fragment of the fusion protein was verified in C3d receptor-expressing Raji cells and flow cytometric analysis. Results: Immunisation of pSG.SS.C3d3.YL.Bin1b recombinant DNA vaccine significantly decreased sperm motility and compromised fertility in male mice. ELISA results showed that the titres of anti-Bin1b IgG in sera of immunised mice increased markedly with the immunisation process. Further, the anti-fertility effect of pSG.SS.C3d3.YL.Bin1b was significantly better than that of pSG.SS.YL.Bin1b DNA vaccine and generated higher titres of anti-Bin1b antibody. Conclusions: Our results show that recombinant DNA vaccine targeting Bin1b can markedly reduce fertility in male mice, providing an alternative approach for birth control.

Objective: To evaluate the contraceptive ability of a synthetic Bin1b peptide in vivo in the rat. Design: Basic research. Setting: University laboratory animal service center. Animal(s): A peptide-based immunization model was developed; rats were injected with the Bin1b specific peptide. Intervention(s): A synthetic peptide segment, MCRSGERKGDICSDP-conjugated with KLH (Bin1b), was used to immunize male wistar rats. Freund's complete adjuvant was used as a control. Main outcome measure(s): Anti-Bin1b levels in sera were evaluated by enzyme-linked immunosorbent assay (ELISA). Anti-Bin1b and control antisera were used to evaluate sperm function inhibition in vitro. The fertility of immunized rats was determined by mating experiment. The testis and epididymides were analyzed by histology. Result(s): Histological studies showed no evidence of orchitis or epididymitis in Bin1b-immunized animals. ELISA results revealed that the titers of anti-Bin1b antibodies in serum increased with the immunization process in immunized rats. Sperm recovered from the corpus epididymidis of the Bin1b-immunized animals exhibited a significant decrease in motility. Immunization of Bin1b also caused a reduction (25%) in fertility after the mating experiment. Conclusion(s): The present study has demonstrated that immunization with Bin1b peptide specifically interferes with sperm motility, resulting in a compromised fertilizing capacity of sperm.