Binding mechanism of affinity ligands for purification of plasmid DNA

Plasmid DNA for therapeutic and vaccination purposes must be highly purified. The high selectivity of affinity chromatography makes it ideal for the isolation of pDNA from complex biological feed stocks. Affinity chromatography makes use of the biological function and/or individual chemical structure of the interacting molecules. However, the success of any affinity purification protocol is dependent on the availability of suitable ligands. In this study, surface plasmon resonance (SPR) based Biacore system has been employed for the detection and quantification of the binding between lac operon (lacO) sequence contained in a pDNA and synthetic peptides based on the DNA-binding domain of the lac repressor protein, lad. The equilibrium dissociation constant (K D) and association and dissociation rate constants (ka, kd) for the interaction between plasmid DNA and designed peptides were determined.

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with unpractically low DNA yields. We have optimized tbe procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 μg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction.

Affinity adsorption of plasmid DNA

The development of a protein-mediated dual functional affinity adsorption of plasmid DNA is described in this work. The affinity ligand for the plasmid DNA comprises a fusion protein with glutathione-S-transferase (GST) as the fusion partner with a zinc finger protein. The protein ligand is first bound to the adsorbent by affinity interaction between the GST moeity and gluthathione that is covalently immobilized to the base matrix. The plasmid binding is then enabled via the zinc finger protein and a specific nucleotide sequence inserted into the DNA. At lower loadings, the binding of the DNA onto the Fractogel, Sepharose, and Streamline matrices was 0.0078 ± 0.0013, 0.0095 ± 0.0016, and 0.0080 ± 0.0006 mg, respectively, to 50 μL of adsorbent. At a higher DNA challenge, the corresponding amounts were 0.0179 ± 0.0043, 0.0219 ± 0.0035, and 0.0190 ± 0.0041 mg, respectively. The relatively constant amounts bound to the three adsorbents indicated that the large DNA molecule was unable to utilize the available zinc finger sites that were located in the internal pores and binding was largely a surface adsorption phenomenon. Utilization of the zinc finger binding sites was shown to be highest for the Fractogel adsorbent. The adsorbed material was eluted with reduced glutathione, and the eluted efficiency for the DNA was between 23% and 27%. The protein elution profile appeared to match the adsorption profiles with significantly higher recoveries of bound GST-zinc finger protein.

Preparation, Analysis and Use of an Affinity Adsorbent for the Purification of GST Fusion Protein

Methods are presented for the preparation, ligand density analysis and use of an affinity adsorbent for the purification of a glutathione S-transferase (GST) fusion protein in packed and expanded bed chromatographic processes. The protein is composed of GST fused to a zinc finger transcription factor (ZnF). Glutathione, the affinity ligand for GST purification, is covalently immobilized to a solid-phase adsorbent (Streamline™). The GST–ZnF fusion protein displays a dissociation constant of 0.6 x10-6 M to glutathione immobilized to Streamline™. Ligand density optimization, fusion protein elution conditions (pH and glutathione concentration) and ligand orientation are briefly discussed.

Plasmid DNA purification via the use of a dual affinity protein

Methods are presented for the production, affinity purification and analysis of plasmid DNA (pDNA). Batch fermentation is used for the production of the pDNA, and expanded bed chromatography, via the use of a dual affinity glutathione S-transferase (GST) fusion protein, is used for the capture and purification of the pDNA. The protein is composed of GST, which displays affinity for glutathione immobilized to a solid-phase adsorbent, fused to a zinc finger transcription factor, which displays affinity for a target 9-base pair sequence contained within the target pDNA. A Picogreen™ fluorescence assay and/or anx ethidium bromide agarose gel electrophoresis assay can be used to analyze the eluted pDNA.

Attributing human uniqueness and human nature to cultural groups : distinct forms of subtle dehumanization

Research on subtle dehumanization has focused on the attribution of human uniqueness to groups (infrahumanization), but has not examined another sense of humanness, human nature. Additionally, research has not extended far beyond Western cultures to examine the universality of these forms of dehumanization. Hence, the attribution of both forms of humanness was examined in three cross-cultural studies. Anglo-Australian and ethnic Chinese attributed values and traits (Study 1, N = 200) and emotions (Study 2, N = 151) to Australian and Chinese groups, and rated these characteristics on human uniqueness and human nature. Both studies found evidence of complementary attributions of humanness for Australians, who denied Chinese human nature but attributed them with greater human uniqueness. Chinese denied Australians human uniqueness, but their attributions of human nature varied for traits, values, and emotions. Study 3 (N = 54) demonstrated similar forms of dehumanization using an implicit method. These results and their implications for dehumanization and prejudice suggest the need to broaden investigation and theory to encompass both forms of humanness, and examine the attribution of both lesser and greater humanness to outgroups.

Age-related changes to adenosine in rat coronary resistance vessels

1. The vasodilator effects of adenosine receptor agonists, isoprenaline and histamine were examined in perfused heart preparations from young (4–6 weeks) and mature (12–20 weeks) rats. 2. Adenosine induced a biphasic concentration-dependent decrease in KCl (35 mM) raised coronary perfusion pressure in hearts from young and mature rats, suggesting the presence of both high- and low-affinity sites for adenosine receptors in the two age groups tested. In heart preparations from mature rats, vasodilator responses to adenosine were significantly reduced compared with responses observed in young rats. 3. Responses to 5′-N-ethylcarboxamidoadenosine (NECA) and 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680) were reduced in preparations from mature rats, whereas the vasodilator actions of N6-cyclopentyladenosine (CPA) and N6-2-(4-aminophenyl)ethyladenosine (APNEA) did not change with age. 4. The results presented in this study suggest that several adenosine receptor subtypes mediate vasodilator responses in the coronary circulation of the rat and that a reduction in response to adenosine with age may be due to changes in the high-affinity receptor site.

The David and Goliath principle: Cultural, ideological, and attitudinal underpinnings of the normative protection of low-status groups from criticism

Two studies documented the “David and Goliath” rule—the tendency for people to perceive criticism of “David” groups (groups with low power and status) as less normatively permissible than criticism of “Goliath” groups (groups with high power and status). The authors confirmed the existence of the David and Goliath rule across Western and Chinese cultures (Study 1). However, the rule was endorsed more strongly in Western than in Chinese cultures, an effect mediated by cultural differences in power distance. Study 2 identified the psychological underpinnings of this rule in an Australian sample. Lower social dominance orientation (SDO) was associated with greater endorsement of the rule, an effect mediated through the differential attribution of stereotypes. Specifically, those low in SDO were more likely to attribute traits of warmth and incompetence to David versus Goliath groups, a pattern of stereotypes that was related to the protection of David groups from criticism.

Heavy vehicle safety in Oman: Police interviews and heavy vehicle driver survey and focus groups. Third Report of the Heavy Vehicle Safety Research in Oman Project for The Research Council of Oman.

Over the past six months the project has undertaken three key, separate, data collection rounds. Each of these rounds focused on essentially different issues within the broader common construct of heavy vehicle road safety. This document will initially report on a series of two key qualitative data collections rounds. Firstly it will detail findings and report on discussions held in focus groups with 43 heavy vehicle drivers. The second qualitative study involved a series of interviews undertaken with 19 police officers from various levels of command and operations within the Royal Oman Police. The final data collection round reported on in this document is a roadside survey questionnaire undertaken with 400 heavy vehicle drivers.

Tailoring surface properties and structure of layered double hydroxides using silanes with different number of functional groups

Four silanes, trimethylchlorosilane (TMCS), dimethyldiethoxylsilane (DMDES), 3-aminopropyltriethoxysilane (APTES) and tetraethoxysilane (TEOS), were adopted to graft layered double hydroxides (LDH) via an induced hydrolysis silylation method (IHS). Fourier transform infrared spectra (FTIR) and 29Si MAS nuclear magnetic resonance spectra (29Si MAS NMR) indicated that APTES and TEOS can be grafted onto LDH surfaces via condensation with hydroxyl groups of LDH, while TMCS and DMDES could only be adsorbed on the LDH surface with a small quantity. A combination of X-ray diffraction patterns (XRD) and 29Si MAS NMR spectra showed that silanes were exclusively present in the external surface and had little influence on the long range order of LDH. The surfactant intercalation experiment indicated that the adsorbed and/or grafted silane could not fix the interlamellar spacing of the LDH. However, they will form crosslink between the particles and affect the further surfactant intercalation in the silylated samples. The replacement of water by ethanol in the tactoids and/or aggregations and the polysiloxane oligomers formed during silylation procedure can dramatically increase the value of BET surface area (SBET) and total pore volumes (Vp) of the products.

Designing hubs for connected learning: social, spatial and technological insights from Coworking, Hackerspaces and Meetup groups

Connected learning, as a design approach, does not restrict learning to a dedicated learning space (school, university, etc.), but considers it to be an aggregation of individual experiences made through intrinsically motivated, active participation in and across various socio-cultural, every-day life environments. Urban places for meeting, interacting and connected learning with people from diverse backgrounds, cultures and areas of expertise are highly significant in the knowledge economy of our 21st century. However, little is yet known about best practices to design and curate such hubs that attract and support interest-driven and socially embedded learning experiences. The research study presented in this paper investigates design aspects that contribute to successful place-based spaces for connected learning. The paper reports findings from observations as well as interviews with users and managers of three different types of local, community-led learning environments, i.e., coworking spaces, hackerspaces, and meetup groups across Australia. The findings reveal social, spatial and technological interventions that these spaces apply to nourish a culture of connected learning, sharing and peer interaction. The discussion suggests a set of design implications for designers, managers and decision makers that have an interest in nourishing a connected learning culture among their user community.

Focusing on what counts: Using exploratory focus groups to enhance the development of an electronic survey in a mixed-methods research design

This paper describes the use of exploratory focus groups to inform the development of a survey instrument in a sequential phase mixed methods study investigating differences in secondary students’ career choice capability. Five focus groups were conducted with 23 year 10 students in the state of New South Wales (NSW), Australia. Analysis of the focus group data informed the design of the instrument for the second phase of the research project: a large-scale cross-sectional survey. In this paper, we discuss the benefits of using sequential phase mixed method approaches when inquiring into complex phenomena such as human capability.