A novel peptide specifically targeting ovarian cancer identified by in vivo phage display

Discovery of peptide ligands that can target human ovarian cancer and deliver chemotherapeutics offers new opportunity for cancer therapy. The advent of phage‐displayed peptide library facilitated the screening of such peptides. In vivo screening that set in a microanatomic and functional context was applied in our study, and a novel peptide WSGPGVWGASVK targeting ovarian cancer was isolated. The phage clone PC3‐1 displaying peptide WSGPGVWGASVK can gain effective access to accumulate in the tumor sites after intravenous injection while reducing its accumulation in normal organs. Positive immunostaining of PC3‐1 was located in both sites of tumor cells and tumor blood vessels, which resulted in a diffuse binding pattern through the tumor. In vitro study results confirmed the capability of peptide WSGPGVWGASVK binding to and being internalized by both tumor cells and angiogenic endothelial cells. Flow cytometry analysis revealed that the peptide bound to SKOV3 cells with Kd value of 5.43 ± 0.4 μM. Taken together, it suggested that peptide WSGPGVWGASVK is a lead candidate for delivering therapeutics to penetrate into tumors. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Construction of an Immunostimulatory Plasmid, pUCpGs10, and Research on its Immune Adjuvant Effect

In order to overcome the instability of CpG ODN in vivo, sequence diversity, and individual differences, eleven CpG ODN fragments were meticulously selected and linked to form a Multi-CpG, which were repeatedly inserted into the cloning vector pUC19 for constructing the recombinant plasmid pUCpGs10 containing ten of Multi-CpG. Using the multi-genotype HCV E1 and multi-epitope complex HCV-T as immunogens, and plasmid pUCpGs10 as the immune adjuvant, Balb/c mice were immunized through nasal and subcutaneous immunization. Strong-specific humoral and cellular immune response were induced, which can obviously inhibit the growth of homograft expressing HCV antigen. The immune adjuvant effect of pUCpGs10 closely matched that of Freund’s complete adjuvant. The plasmid pUCpGs10 can significantly improve IgA content in serum and different mucosal extract and systematical T-cell response via intranasal immunization. In conclusions, the newly constructed immunostimulatory plasmid pUCpGs10 is able to effectively activate the humoral and cellular immune activity, and possesses activation on mucosal immune response.     

CODON OPTIMIZATION INCREASES HUMAN KALLISTATIN EXPRESSION IN Escherichia coli

A unique serpin, kallistatin, displays vasodilatory, antiangiogenic, anti-inflammatory, and antioxidant activity. Difficulty and low efficacy of obtaining recombinant kallistatin limit the wide investigation of its biological and pathological function. The present study employed a codon optimization algorithm to redesign the kallistatin gene and achieved a high yield of recombinant kallistatin protein. The kallistatin codons were redesigned for a more suitable Escherichia coli host without altering amino acids. Base composition and GC% content were compared between synthetic optimized kallistatin (opti-kallistatin) and wild-type kallistatin (wt-kallistatin). Both opti-kallistatin and wt-kallistatin were purified using Ni-NTA His-binding resins through fast protein liquid chromatography (FPLC). The identity and purity of kallistatin were confirmed by Coomassie blue staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot analysis. The output of opti-kallistatin protein was ～2-fold increase (2.09 ± 0.23 mg/L) compared to wt-kallistatin (1.05 ± 0.2 mg/L). These results suggest that more common codon optimization in the E. coli host significantly increases the yield of heterologous human protein yields. This approach will remarkably facilitate the further investigation of kallistatin in vitro and in vivo.     

Expression,purification, and characterization of N-terminal His-tagged proteins with mutations in zinc finger 3 of zinc finger protein ZNF191(243–368)

Abstract

Zinc finger protein ZNF191(243–368), the zinc finger region of ZNF191, is potentially associated with cell proliferation in hepatocellular carninoma. A His-tag expression system was used to express and purify proteins with mutations in the zinc finger 3 of ZNF191(243–368) for analysis of protein properties, structure, and functions. The purification of the His-tag fusion proteins was simpler and faster than that of the ZNF191(243–368) inclusion bodies. The properties and structures of the His-tag fusion mutant proteins were investigated using spectrographic techniques and DNA hydrolysis experiment. The His₆-tag system could be used to express ZNF191(243–368). The presence of the His₆-tag at the N-terminus of ZNF191(243–368) did not evidently affect its properties and structure. However, the site-directed mutations in zinc finger 3 affected the structure of the protein. The DNA hydrolase activity of His₆-ZF-F3/H4 suggested that four histidines in zinc finger 3 might form a structure similar to that of the active center in a hydrolase. This work reports that continuous histidines need to form a certain structure for specific functions, and provides new insights into the design of an artificial nuclease.     

Production of γ-aminobutyric acid in Escherichia coli by engineering MSG pathway

Abstract

The compound γ-aminobutyric acid (GABA) has many important physiological functions. The effect of glutamate decarboxylases and the glutamate/GABA antiporter on GABA production was investigated in Escherichia coli. Three genes, gadA, gadB, and gadC were cloned and ligated alone or in combination into the plasmid pET32a. The constructed plasmids were transformed into Escherichia coli BL21(DE3). Three strains, E. coli BL21(DE3)/pET32a-gadA, E. coli BL21(DE3)/pET32a-gadAB and E. coli BL21(DE3)/pET32a-gadABC were selected and identified. The respective titers of GABA from the three strains grown in shake flasks were 1.25, 2.31, and 3.98?g/L. The optimal titer of the substrate and the optimal pH for GABA production were 40?g/L and 4.2, respectively. The highest titer of GABA was 23.6?g/L at 36?h in batch fermentation and was 31.3?g/L at 57?h in fed-batch fermentation. This study lays a foundation for the development and use of GABA.     

Comprehensive annotation of microRNA expression profiles

Background

MicroRNAs (miRNAs) regulate many biological processes by post-translational gene silencing. Analysis of miRNA expression profiles is a reliable method for investigating particular biological processes due to the stability of miRNA and the development of advanced sequencing methods. However, this approach is limited by the broad specificity of miRNAs, which may target several mRNAs.

Result

In this study, we developed a method for comprehensive annotation of miRNA array or deep sequencing data for investigation of cellular biological effects. Using this method, the specific pathways and biological processes involved in Alzheimer’s disease were predicted with high correlation in four independent samples. Furthermore, this method was validated for evaluation of cadmium telluride (CdTe) nanomaterial cytotoxicity. As a result, apoptosis pathways were selected as the top pathways associated with CdTe nanoparticle exposure, which is consistent with previous studies.

Conclusions

Our findings contribute to the validation of miRNA microarray or deep sequencing results for early diagnosis of disease and evaluation of the biological safety of new materials and drugs.