Design of bacterial polyester beads for recombinant protein production, biomolecule separation and detection : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

Protein recovery and biomolecule detection are commonly required for scientific research as well as industrial activities. However, it is generally complicated and costly either to produce and purify recombinant proteins (especially therapeutic proteins) from engineered Escherichia coli cells, or to directly separate proteins or detect other biomolecules from natural sources. Here the PHA synthase (PhaC) mediated polyhydroxyalkanoate (PHA) bead display technology was explored as a solution to these problems by developing streamlined processes with less complex steps to achieve protein recovery and biomolecule detection. Firstly, by fusing a target protein to PhaC via a self-cleavable linker tag of either sortase (sortase A from Staphylococcus aureus) or intein (DnaB mini intein from Synechocystis sp. PCC 6803), new self-cleavable recombinant protein production and purification resins were developed. It was shown that the PhaC fusion could mediate in vivo production of PHA beads displaying the target protein. Functional target protein could be obtained at high purity from isolated PHA beads by incubation with CaCl2 and triglycine (in the case of the self-cleavable sortase tag) or by a pH shift to 6 (in the case of the self-cleavable intein tag). Six recombinant proteins were successfully produced and purified via the intein approach, including 3 model proteins (Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, and the synthetic immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus) and 3 therapeutic proteins (human tumour necrosis factor alpha (TNFα), human interferon alpha-2b (IFNα2b), and human granulocyte colony-stimulating factor (G-CSF)). Of these, TNFα and IFNα2b were also successfully produced and purified via the sortase approach. Secondly, in vivo one-step production of PHA affinity resins was achieved by fusing to PhaC differently customised OBody ligands. These ligands were previously engineered by other groups from the OB-fold domain of aspartyl-tRNA synthetase (aspRS) from Pyrobactulum aerophilum, by using phage display technology, to have specific binding affinities to biomolecules of interest. The resulting recombinant OBody beads were used for lysozyme sepration from a complex substrate, and for progesterone (P4) binding. Further optimisation of the P4 binding condition is necessary before the OBody bead system can be used for P4 detection in bovine milk. However, recombinant immobilisation of OBody ligands on the surface of PHA beads expands not only the attractiveness of these emerging OBody scaffolds, but also the utility scope of PHA beads as affinity resins

Mortality prediction and influencing factors for intensive care unit patients with acute tubular necrosis: random survival forest and cox regression analysis

Background: Patients with acute tubular necrosis (ATN) not only have severe renal failure, but also have many comorbidities, which can be life-threatening and require timely treatment. Identifying the influencing factors of ATN and taking appropriate interventions can effectively shorten the duration of the disease to reduce mortality and improve patient prognosis.Methods: Mortality prediction models were constructed by using the random survival forest (RSF) algorithm and the Cox regression. Next, the performance of both models was assessed by the out-of-bag (OOB) error rate, the integrated brier score, the prediction error curve, and area under the curve (AUC) at 30, 60 and 90 days. Finally, the optimal prediction model was selected and the decision curve analysis and nomogram were established.Results: RSF model was constructed under the optimal combination of parameters (mtry = 10, nodesize = 88). Vasopressors, international normalized ratio (INR)_min, chloride_max, base excess_min, bicarbonate_max, anion gap_min, and metastatic solid tumor were identified as risk factors that had strong influence on mortality in ATN patients. Uni-variate and multivariate regression analyses were used to establish the Cox regression model. Nor-epinephrine, vasopressors, INR_min, severe liver disease, and metastatic solid tumor were identified as important risk factors. The discrimination and calibration ability of both predictive models were demonstrated by the OOB error rate and the integrated brier score. However, the prediction error curve of Cox regression model was consistently lower than that of RSF model, indicating that Cox regression model was more stable and reliable. Then, Cox regression model was also more accurate in predicting mortality of ATN patients based on the AUC at different time points (30, 60 and 90 days). The analysis of decision curve analysis shows that the net benefit range of Cox regression model at different time points is large, indicating that the model has good clinical effectiveness. Finally, a nomogram predicting the risk of death was created based on Cox model.Conclusion: The Cox regression model is superior to the RSF algorithm model in predicting mortality of patients with ATN. Moreover, the model has certain clinical utility, which can provide clinicians with some reference basis in the treatment of ATN and contribute to improve patient prognosis

Duck Eggs and Black-Yolked Salted Duck Eggs: Bacterial Diversity on Eggshells and Gene Function Prediction Using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt)

After comparing the bacterial load of black-yolked salted duck eggs (BSE) and that of duck eggs with different cleanliness degrees, it was found that the total bacterial count on the shell of severely stained eggs (DE) was distinctly higher than that on the shell of non-visually stained eggs (CE), and the total bacterial count in the shell membrane and contents of BSE increased greatly. Furthermore, 16S rDNA gene sequencing was used to analyze the differences in the composition and abundance of bacterial communities in CE, DE and BSE. It turned out that Proteobacteria, Firmicutes and Actinobacteria were the dominant phyla, accounting for 34.35%, 27.25% and 17.28% of the total abundance, respectively, while Bacteroidetes accounted for 8.29% of the total abundance. Proteobacteria was the dominant bacteria in DE and accounted for 89.01% of the total abundance, while the relative abundance of Firmicutes and Actinobacteria were 6.16% and 3.98%. The dominant phyla in BSE were Proteobacteria (76.50%), Bacteroidetes (8.00%), Actinobacteria (6.76%) and Firmicutes (5.43%). The abundance of Proteobacteria in DE and BSE was significantly increased. The dominant bacterial genera in CE were Nesterdella (9.08%), Campylobacter (7.91%), Streptococcus (3.41%) and Oligomonas (2.92%). In DE, Psychrobacter was the dominant genus, accounting for 86.01% of the total abundance, and the relative abundance of Acinetobacter was 2.20%. The dominant bacteria in BSE were Rolstonia (22.91%), Serratia (5.05%) and Actinomycetes (3.32%). The cluster analysis of bacterial diversity showed that the dominant microorganisms of BSE were close to those of DE. The microbial phenotype analysis showed that the dominant microorganisms of BSE were Gram-negative bacteria, had high oxidative stress tolerance and pathogenicity, and could tolerate oxidizing detergents. The results of microbial gene function prediction and species composition of metabolic pathways demonstrated that bacterial carbohydrate decomposition, lipid transport and metabolic oxidation in DE were stronger than those in CE. More coenzyme transport and metabolic pathways could make the biochemical reactions of bacteria from DE faster to produce more secondary metabolites, thus resulting in changes in the quality of duck eggs. This is consistent with the highest bacterial abundance in the secondary metabolite biosynthesis, transport and catabolism pathways in BSE. The results of this study provide theoretical support for the cleaner production of duck eggs

Tin Oxide Nanorod Array-Based Electrochemical Hydrogen Peroxide Biosensor

SnO2 nanorod array grown directly on alloy substrate has been employed as the working electrode of H2O2 biosensor. Single-crystalline SnO2 nanorods provide not only low isoelectric point and enough void spaces for facile horseradish peroxidase (HRP) immobilization but also numerous conductive channels for electron transport to and from current collector; thus, leading to direct electrochemistry of HRP. The nanorod array-based biosensor demonstrates high H2O2 sensing performance in terms of excellent sensitivity (379 μA mM−1 cm−2), low detection limit (0.2 μM) and high selectivity with the apparent Michaelis–Menten constant estimated to be as small as 33.9 μM. Our work further demonstrates the advantages of ordered array architecture in electrochemical device application and sheds light on the construction of other high-performance enzymatic biosensors

Anticancer activity of an extract from needles and twigs of Taxus cuspidata and its synergistic effect as a cocktail with 5-fluorouracil

<p>Abstract</p> <p>Background</p> <p>Botanical medicines are increasingly combined with chemotherapeutics as anticancer drug cocktails. This study aimed to assess the chemotherapeutic potential of an extract of <it>Taxus cuspidata </it>(<it>TC</it>) needles and twigs produced by artificial cuttage and its co-effects as a cocktail with 5-fluorouracil (5-FU).</p> <p>Methods</p> <p>Components of <it>TC </it>extract were identified by HPLC fingerprinting. Cytotoxicity analysis was performed by MTT assay or ATP assay. Apoptosis studies were analyzed by H & E, PI, TUNEL staining, as well as Annexin V/PI assay. Cell cycle analysis was performed by flow cytometry. 5-FU concentrations in rat plasma were determined by HPLC and the pharmacokinetic parameters were estimated using 3p87 software. Synergistic efficacy was subjected to median effect analysis with the mutually nonexclusive model using Calcusyn1 software. The significance of differences between values was estimated by using a one-way ANOVA.</p> <p>Results</p> <p><it>TC </it>extract reached inhibition rates of 70-90% in different human cancer cell lines (HL-60, BGC-823, KB, Bel-7402, and HeLa) but only 5-7% in normal mouse T/B lymphocytes, demonstrating the broad-spectrum anticancer activity and low toxicity to normal cells of <it>TC </it>extract <it>in vitro</it>. <it>TC </it>extract inhibited cancer cell growth by inducing apoptosis and G₂/M cell cycle arrest. Most interestingly, <it>TC </it>extract and 5-FU, combined as a cocktail, synergistically inhibited the growth of cancer cells <it>in vitro</it>, with Combination Index values (CI) ranging from 0.90 to 0.26 at different effect levels from IC50 to IC90 in MCF-7 cells, CI ranging from 0.93 to 0.13 for IC40 to IC90 in PC-3M-1E8 cells, and CI < 1 in A549 cells. In addition, the cocktail had lower cytotoxicity in normal human cell (HEL) than 5-FU used alone. Furthermore, <it>TC </it>extract did not affect the pharmacokinetics of 5-FU in rats.</p> <p>Conclusions</p> <p>The combinational use of the <it>TC </it>extract with 5-FU displays strong cytotoxic synergy in cancer cells and low cytotoxicity in normal cells. These findings suggest that this cocktail may have a potential role in cancer treatment.</p

In vivo polyester immobilized sortase for tagless protein purification

Background: Laboratory scale recombinant protein production and purification techniques are often complicated, involving multiple chromatography steps and specialized equipment and reagents. Here it was demonstrated that recombinant proteins can be expressed as covalently immobilized to the surface of polyester (polyhydroxyalkanoate, PHA) beads in vivo in Escherichia coli by genetically fusing them to a polyester synthase gene (phaC). The insertion of a self-cleaving module, a modified sortase A (SrtA) from Staphylococcus aureus and its five amino acid recognition sequence between the synthase and the target protein led to a simple protein production and purification method. Results: The generation of hybrid genes encoding tripartite PhaC-SrtA-Target fusion proteins, enabled immobilization of proteins of interest to the surface of PHA beads in vivo. After simple cell lysis and isolation of the PHA beads, the target proteins could be selectively and efficiently released form the beads by activating the sortase with CaCl2 and triglycine. Up to 6 mg/l of soluble proteins at a purity of ~98 % could be isolated in one step with no optimization. This process was used to produce and isolate three proteins: Green fluorescent protein, maltose binding protein and the Mycobacterium tuberculosis vaccine candidate Rv1626. Conclusions: We have developed a new technique for easy production and purification of recombinant proteins. This technique is capable of producing and purifying high yields of proteins suitable for research application in less than 2 days. No costly or specialized protein chromatography equipment, resins, reagents or expertise are required.Full Tex

Factors Influencing Polyol Liquefaction of Nut Shells of Different Camellia Species

The liquefaction rates and kinetics of nut shells of different Camellia species in PEG400/glycerol/H2SO4 liquefying solvent were investigated. Changes in major components including cellulose, hemicellulose, and lignin as well as cellulose crystallinity of the nut shells were determined. The compositions of the liquefaction residues were analyzed. Results indicated that, under the same conditions, the liquefaction rates of nut shells of different Camellia species were noticeably different and the PEG400/glycerol/H2SO4 liquefaction agent was not suitable for the liquefaction of the nut shells of all Camellia species. The burst liquefaction of Camellia nut shells (CNSs) that occurred during the first stage was due to the rapid degradation of hemicellulose, acid-soluble lignin, and amorphous cellulose. The liquefaction during the second stage became very slow, mostly because the swelling and decomposition of crystalline cellulose was very difficult to achieve with the liquefying agent and the liquefaction products inhibited liquefaction at later stages. The liquefaction residues of CNSs were composed of crystalline cellulose, small amounts of hemicellulose, acid-insoluble lignin, and ash. Ash was partially dissolved in the liquefying agent. The liquefaction rates of all CNSs tested in this study showed linear relationships with time, with coefficients of determination (R2) greater than 0.7082, indicating that the liquefaction of CNS was a pseudo-first-order reaction

Hsp90 Interacts With Tm-22 and Is Essential for Tm-22-Mediated Resistance to Tobacco mosaic virus

The tomato resistance gene Tm-22 encodes a coiled coil-nucleotide binding site-leucine rich repeat type resistance protein and confers effective immune response against tobamoviruses by detecting the presence of viral movement proteins (MPs). In this study, we show that the Nicotiana benthamiana Heat shock protein 90-kD (Hsp90) interacts with Tm-22. Silencing of Hsp90 reduced Tm-22-mediated resistance to Tobacco mosaic virus (TMV) and the steady-state levels of Tm-22 protein. Further, Hsp90 associates with SGT1 in yeast and in plant cells. These results suggest that Hsp90-SGT1 complex takes part in Tm-22-mediated TMV resistance by functioning as chaperone to regulate Tm-22 stability