Cell-Free Display Techniques for Protein Evolution.

Cell-free protein synthesis (CFPS) with flexibility and controllability can provide a powerful platform for high-throughput screening of biomolecules, especially in the evolution of peptides or proteins. In this chapter, the emerging strategies for enhancing the protein expression level using different source strains, energy systems, and template designs in constructing CFPS systems are summarized and discussed in detail. In addition, we provide an overview of the ribosome display, mRNA display, cDNA display, and CIS display in vitro display technologies, which can couple genotype and phenotype by forming fusion complexes. Moreover, we point out the trend that improving the protein yields of CFPS itself can offer more favorable conditions for maintaining library diversity and display efficiency. It is hoped that the novel CFPS system can accelerate the development of protein evolution in biotechnological and medical applications.

Efficient In Vitro Full-Sense-Codons Protein Synthesis.

Termination of translation is essential but hinders applications of genetic code engineering, e.g., unnatural amino acids incorporation and codon randomization mediated saturation mutagenesis. Here, for the first time, it is demonstrated that E. coli Pth and ArfB together play an efficient translation termination without codon preference in the absence of class-I release factors. By degradation of the targeted protein, both essential and alternative termination types of machinery are completely removed to disable codon-dependent termination in cell extract. Moreover, a total of 153 engineered tRNAs are screened for efficient all stop-codons decoding to construct a codon-dependent termination defect in vitro protein synthesis with all 64 sense-codons, iPSSC. Finally, this full sense genetic code achieves significant improvement in the incorporation of distinct unnatural amino acids at up to 12 positions and synthesis of protein encoding consecutive NNN codons. By decoding all information in nucleotides to amino acids, iPSSC may hold great potential in building artificial protein synthesis beyond the cell.

Targeting HSF1 leads to an antitumor effect in human epithelial ovarian cancer.

Late diagnosis and lack of specific therapeutic targets contribute to the low survival rate of patients with epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy. Therefore, the screening of diagnostic markers and the identification of therapeutic targets are urgently required. Heat shock factor 1 (HSF1) has been demonstrated to be overexpressed in certain malignancies and to be involved in tumor initiation, development, transformation and metastasis. It is believed that HSF1 is a promising candidate for antitumor therapy. However, its expression pattern and function in ovarian cancer are far from being fully elucidated. Therefore, we examined the HSF1 expression in human EOC tissues, and evaluated its carcinogenesis-promoting activity in a xenograft tumor model. Examination of HSF1 expression in human EOC tissues was performed by immunohistochemical assay using ovarian tissue blots. Specific short hairpin RNA (shRNA) against HSF1 was employed to knockdown HSF1 in SKOV3 cells. Cell proliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay; cell cycle distribution and apoptosis were determined by flow cytometric analysis. In normal ovarian tissues, HSF1 was barely detected, whereas, high expression of HSF1 was found in malignant EOC tissues, including serous, mucinous, endometrioid, and clear cell EOC tissues. Suppressed proliferative activity and intensified apoptosis were observed in HSF1-knockdown SKOV3 cells. In nude mouse xenografts, downregulation of HSF1 was found to cause reduced carinogenesis, indicating the antitumor effect induced by modulation of HSF1 against EOC. Our findings suggest that HSF1 may be considered as a potential candidate diagnostic marker of human EOC, and that modulation of HSF1 could be a promising therapeutic strategy against human EOC.

Study on preparation and separation of Konjac oligosaccharides.

To study the preparation and separation of Konjac oligosaccharides, Konjac Glucomannan was degraded by the combination of γ-irradiation and ß-mannanase, and then the degradation product was separated by ultrafiltration. To our interest, for most of Konjac oligosaccharides obtained by this method, the molecular mass was lower than 2200 Da. In addition, the 1000 Da molecular weight cut off membrane could effectively separate the Konjac oligosaccharides. In conclusion, the combination of γ-irradiation and ß-mannanase was an efficient method to obtain Konjac oligosaccharides, and the oligosaccharides of molecular mass lower than 1000 Da could be effectively separated by ultrafiltration.

Synthesis and characterization of konjac glucomannan-graft-polyacrylamide via gamma-irradiation.

The synthesis of konjac glucomannan-graft-polyacrylamide (KGM-g-PAM) was carried out at 25 degrees C by gamma-irradiation under a N2 atmosphere. The effects of absorbed radiation dosage and monomer concentration on grafting yield and water absorbency were studied. The grafted copolymers were characterized using Fourier Transform Infrared(FTIR) spectroscopy, nuclear magnetic resonance (NMR), x-ray diffraction (XRD),thermogravimetric analysis (TGA) and gel permeation chromatography (GPC). The grafting yield was observed to increase with increasing absorbed dosage and monomer concentration. Compared with the original KGM, the grafted copolymers exhibited better thermal stability and water absorbency. The results suggest that gamma-irradiation is convenient and efficient for inducing graft copolymerization of KGM and acrylamide (AM).

Purification and properties of Bacillus subtilis SA-22 endo-1, 4-beta-D-mannanase.

beta-mannanase (EC 3.2.1.78) from Bacillus subtilis SA-22 was purified successively by ammonium sulfate precipitation, hydroxyapatite chromatography, Sephadex G-75 gel filtration and DEAE-52 anion-exchange chromatography. Through these steps, the enzyme was concentrated 30.75-fold with a recovery rate of 23.43%, with a specific activity of 34780.56 u/mg. Molecular weight of the enzyme was determined to be 38 kD by SDS-PAGE and 34 kD by gel filtration. The results revealed that the optimal pH value for the enzyme was 6.5 and the optimal temperature was 70 degrees C. The enzyme is stable between pH 5 to 10. The enzyme remained most of its activity after a treatment of 4 h at 50 degrees C, but lost 25% of activity at 60 degrees C for 4 h, lost 50% of activity at 70 degrees C for 3 h. The enzyme activity was strongly inhibited by Hg2+. The Michaelis constants (Km) were measured as 11.30 mg/mL for locust bean gum and 4.76 mg/mL for konjac powder, while Vmax for these two polysaccharides were 188.68 (micromol x mL(-1) x min(-1)) and 114.94 (micromol x mL(-1) x min(-1)), respectively.