Effect of salt on purification of plasmid DNA using size-exclusion chromatography

In the present study, we compared the performances of size-exclusion chromatography for the purification of plasmid DNA when different concentrations (0.5M, 1M, 2M, respectively) of two types of salt (NaCl and (NH(4))(2)SO(4)) are present in running buffers. Our experiment results displayed that it is not only the resolution of RNA but also those of supercoiled plasmid DNA and host's genomic DNA were increased greatly in the presence of high concentration of water-structure salt. We deduce that two separation modes may be involved in the process: The supercoiled plasmid DNA is influenced mainly by compaction effect and eluted in the size-exclusion mode; whereas, RNA and genomic DNA are influenced mainly by hydrophobic effect due to their stretched and loose structures and eluted in the interaction mode. This method led to an improved efficiency of size-exclusion chromatography.     

巯基吡啶配基液相色谱固定相的制备及其对质粒的快速纯化

在基因治疗中,质粒是一种常用的非病毒型载体。由于目前市场上对质粒的需求量很大,因此有必要开发大规模的质粒制备技术。该文以双孔型流通色谱介质为基质,以巯基吡啶为配基,制得了液相色谱固定相,并考察了其对质粒DNA的纯化效果,分析了分离机理。结果表明:该分离介质对质粒的纯化是基于疏水作用;当上样量为10 mL (质粒质量浓度为0.30 mg/mL)、流速高达4 mL/min时,质粒仍然能以100%的收率实现纯化,其纯度为100%。该色谱介质对RNA的柱容量高达2.2 mg/mL。     

Clearance of host cell impurities from plasmid-containing lysates by boronate adsorption

The ability of boronate adsorption to clear Escherichia coli impurities directly from plasmid-containing lysates (∼pH 5.2) was evaluated. Results show that 3-aminophenyl boronate (PB) controlled pore glass (CPG) is able to adsorb not only those species that bear cis-diol groups (RNA, lipopolysaccharides-LPS), and are thus able to form covalent bonds with boronate, but also cis-diol-free proteins and genomic DNA (gDNA) fragments, while leaving most plasmid DNA in solution. Control runs performed with phenyl Sepharose and with PB-free CPG beads ruled out hydrophobic interactions with the phenyl ring and non-specific interactions with the glass matrix, respectively, as being responsible for RNA and gDNA adsorption. In batch mode, up to 97.6 ± 3.1% of RNA, 94.6 ± 0.8% of proteins and 96.7 ± 11.7% of gDNA were cleared after 30 min, with a plasmid yield of 64%. In fixed-bed mode, most of the plasmid was recovered in the flowthrough (96.2 ± 4.0%), even though the RNA (65.5 ± 2.8%), protein (84.4 ± 1.3%) and gDNA clearance (44.7 ± 14.1%) were not as effective. In both cases, the LPS content was removed to a residual value of less than 0.005 EU/ml. The method is fast and straightforward, circumvents the need for pre-treatment of the feed and may contribute to shorten plasmid purification processes, as the treated streams can proceed directly to the final polishing steps.     

聚丙烯熔体自干扰流动对其凝聚态取向效应的影响

通过一种由两个浇口小流道共享一个分流道的新型浇口 ,使两股熔体流入型腔 ,并在型腔内产生自干扰流动 (SIF) .在平行于注射方向熔体会产生迭加效应 ,而在垂直于注射方向熔体会产生挤压作用 ,并形成横向流动 .探讨了这种自干扰流动对注射PP试样的取向效应的影响 .采用热收缩和X 射线衍射法分别对分子链取向和晶体取向进行了检测 .热收缩试验结果显示SIF样比常规流动 (CFP)试样具有较小的链取向 .透射X 射线衍射的检测结果表明SIF样和CFP样在剪切层晶体具有大的取向效应 ,SIF样晶体的取向度为 65 % ,而CFP样则为 79% .     

Investigation of DNA condensing properties of amphiphilic triblock cationic polymers by atomic force microscopy

Introduction of nucleic acids into cells is an important biotechnology research field which also holds great promise for therapeutic applications. One of the key steps in the gene delivery process is compaction of DNA into nanometric particles. The study of DNA condensing properties of three linear cationic triblock copolymers poly(ethylenimine-b-propylene glycol-b-ethylenimine), namely, LPEI(50)-PPG(36)-LPEI(50), LPEI(19)-PPG(36)-LPEI(19), and LPEI(14)-PPG(68)-LPEI(14), indicates that proper DNA condensation is driven by both the charge and the size of the respective cationic hydrophilic linear polyethylenimine (LPEI) and neutral hydrophobic poly(propylene glycol) (PPG) parts. Atomic force microscopy was used to investigate the interactions of the triblock copolymers with plasmid DNA at the single molecule level and to enlighten the mechanism involved in DNA condensation.     

The use of capillary electrophoresis with entangled polymer matrix to analyse plasmid DNA and a cationic lipid/cholesterol liposome: DNA complex

Summary A liposome based gene therapeutic product is being developed for the treatment of cystic fibrosis. The product comprises a complex of plasmid DNA and a cationic lipid/cholesterol liposome. Using CE with an entangled polymer matrix, routine separations of linear, supercoiled and open-circle conformers of DNA in plasmid DNA and the liposome complex have been performed. The CE method has been used to support novel quality control and process validation for the manufacture of plasmid DNA and to monitor the degradation of DNA in the liposome complex. Significant features of the method are simple sample preparation and the use of direct UV detection, avoiding the use of potentially mutagenic reagents.     

Adsorption of DNA and dodecyl trimethylammonium bromide mixtures at the air/water interface: a neutron reflectometry study

The interactions between dodecyl trimethylammonium bromide (C12TAB) and two samples of DNA with widely differing molecular weights have been studied using surface tension and neutron reflectometry. Neutron reflection data show that the surfactant and polymer are adsorbed together in a highly cooperative fashion over a 1000-fold change in surfactant concentration. Furthermore, the shorter DNA fragments adsorb with C12TAB as trilayers at higher surfactant concentrations, with overall layer thicknesses of 65-70 A. The high molecular weight DNA, however, shows only approximate monolayer adsorption with thicknesses varying from 19 to 26 A over the entire range of C12TAB concentrations. The difference in behavior between the different samples is believed to be a result of the rigid double helical structure of DNA which makes the formation of bulk phase polymer/micelle aggregates much less favorable for the short fragments. The resulting increase in the critical aggregation concentration (CAC) then leads to the adsorption of additional surfactant/polymer complex to the underside of the initial stable surface active DNA/C12TAB complex. Comparison with previous results obtained for synthetic polyelectrolytes shows that DNA/C12TAB complexes are not capable of reducing the surface tensions to the extent that other mixtures such as the poly(styrene sulfonate)/C12TAB mixtures do. A possible reason for this is the high rigidity of DNA combined with the fact that its hydrophobic moieties are positioned within the double helix so that the external molecule is largely hydrophilic.     

Water-repellent acylated and silylated wood samples and their surface analytical characterization

The interest for chemical modification of wood with the aim of preserving it from physical and biochemical deterioration is rapidly increasing. Among the possibilities, acetylation has a great potential for improving the dimension instability of wood against moisture and the durability of wooden objects. Recently, a new acetylating agent, isopropenyl acetate has attracted attention giving an improved acetylating method. By increasing the size of the acyl group, a positive effect on the wood hydrophobicity can be reached. The anhydrides, however, are less reactive. Another applicable surface modification is the silylation of wood that renders the wood surface hydrophobic.

In this paper, we report about the acylation and silylation of different wood samples. A new manufacturing process is tested in which low-value Eucalyptus wood is mixed acylated. The reagent mixture contains at least two kinds of acyl group. One of them is acetyl group; the other(s) can be user-defined. We also report about a silylation method, in which catalyst facilitates the chemisorption process. The extent of modification of the treated wood samples was evaluated by means of weight percent gain, ATR-FTIR, and pyrolysis-gas chromatography–mass spectrometry (Py-GC/MS). Methylene blue adsorption of selected samples was also studied. The result of analytical measurements was in agreement with that of wettability studies.     

Gel network photodisruption: a new strategy for the codelivery of plasmid DNA and drugs

In the last 5 years, we have gained further insight on the physical/chemical field of DNA gels. Our expertise on the gel swelling behavior, compaction of DNA by cationic entities, as lipids and surfactants, as well as on the assembly structures of these complexes allow us for the development of novel systems to be used in a variety of biomedical applications. In our previous reports, the physicochemical characterization has been well-established, and now one can evolve to the challenge of using DNA-based carriers in the biological area. Moreover, a new plasmid DNA (pDNA) hydrogel that is porous, is able to swell in the presence of additives, is biocompatible and, thus, is suitable to be used therapeutically was prepared. Here, the dual release of pDNA and solutes with pharmaceutical interest was the main challenge, and thus, we report on the photodisruption of plasmid DNA (pDNA) gels cross-linked with ethylene glycol diglycidyl ether (EGDE) as a strategy for this simultaneous release. The disruption over time, after the irradiation of the gel with ultraviolet light (400 nm), was characterized through the cumulative plasmid DNA release, the evolution in dry weight, the extent of swelling, and also the variations in the gel mesh size. The controlled release of different molecular weight solutes from plasmid DNA gels was investigated, and the influence of both the hydrogel degradation and cross-linker density on the release kinetics were addressed. While the release of lysozyme follows a Fickian process, the release of bovine serum albumin (BSA) and fluoresceinisothiocyanato-dextran (FITC-dextran) is characteristic of a Super Case II release phenomena. In addition, the size of the three solutes partially influences the release behavior; polymer chain mobility and the degree of swelling also play a role. To gain a fundamental understanding of drug release profile from pDNA matrices, in vitro release studies were evaluated using several anti-inflammatory drugs. The quantification of the release mechanism indicates a Super Case II release profile, which can be related with the gel swelling degree. A correlation between the drug release trend and the drug hydrophobicity can be found, with more hydrophobic drugs showing a slower release rate. In brief, this new pDNA gel system is biocompatible, is degradable upon light irradiation, and allows for the controlled and sustained release of plasmid DNA and incorporated solutes. This codelivery of pDNA and drugs would find relevant clinical uses due to the possibility of gene and nongene therapy combination in order to improve the therapeutic efficiency.     

Integrated process for purification of plasmid DNA using aqueous two-phase systems combined with membrane filtration and lid bead chromatography

An integrated process for purifying a 6.1 kilo base pair (kbp) plasmid from a clarified Escherichia coli cell lysate based on an ultra/diafiltration step combined with polymer/polymer aqueous two-phase system and a new type of chromatography is described. The process starts with a volume reduction (ultrafiltration) and buffer exchange (diafiltration) of the clarified lysate using a hollow fibre membrane system. The concentrated and desalted plasmid solution is then extracted in a thermoseparating aqueous two-phase system, where the contaminants (RNA and proteins) to a large extent are removed. While the buffer exchange (diafiltration) is necessary in order to extract the plasmid DNA exclusively to the top phase, experiments showed that the ultrafiltration step increased the productivity of the aqueous two-phase system by a factor of more than 10. The thermoseparated water phase was then subjected to a polishing step using lid bead chromatography. Lid beads are a new type of restricted access chromatography beads, here with a positively charged inner core that adsorbed the remaining RNA while its inert surface layer prevented adsorption of the plasmid DNA thus passing in the flow-through of the column. Differently-sized plasmid DNA in the range of 2.7-20.5 kbp were also partitioned in the aqueous two-phase system. Within this size range, all plasmid DNA was exclusively extracted to the top phase. The complete process is free of additives and easy scalable for use in large scale production of plasmid DNA. The overall process yield for plasmid DNA was 69%.     

DNA spike studies for demonstrating improved clearance on chromatographic media

DNA spike clearance methods were used to demonstrate improved clearance factors on anion exchange and hydrophobic interaction columns used in the production of human therapeutic proteins. DNA clearance at large-scale was first measured for a monoclonal antibody expressed in Chinese Hamster Ovary (CHO) cells and an antibody fragment expressed in Escherichia coli. Small-scale spike experiments were then performed on individual chromatographic steps using host-specific DNA paired with TaqMan PCR assay methods. This approach has advantages of improved specificity, sensitivity, cost and throughput compared to other types of spike clearance methods. The anion exchange column used in the monoclonal antibody process was shown to have very high capacity for CHO DNA, resulting in greater than 7.1 log reduction. The anion exchange and hydrophobic interaction columns used in the antibody fragment process were shown to have high E. coli DNA clearance capability, with greater than 5.1 and 5.3 logs clearance, respectively. Compared to the large-scale process, higher log reduction values were achieved in small-scale spike clearance studies by challenging the chromatographic steps with load DNA levels 2–5 logs higher than the large-scale process levels. Using highly specific and sensitive spike clearance methods, we demonstrated consistently high DNA clearance factors for each of the production processes that meet industry and regulatory standards for human therapeutics. The method is applicable to a broad range of industrial scale processes where demonstration of the robustness of DNA clearance is necessary to support development or licensure of biopharmaceutical products.     

Analysis of human tear proteins by different high-performance liquid chromatographic techniques

A comparison of the efficiencies of hydrophobic interaction chromatography, ion-exchange chromatography, reversed-phase chromatography and gel permeation chromatography in the separation of tear proteins was made using a variety of different buffers. Separation of immunoglobulins, lactoferrin, albumin, PMFA (protein migrating faster than albumin) and lysozyme was accomplished by gel permeation chromatography in less than 30 min using a TSK-type SW3000 column equilibrated with ammonium acetate buffer (pH 4.1) with a high reproducibility. When gel permeation chromatography was used as a completely automated diagnostic method, only minute volumes (1.0 microliter) of tear samples were necessary for the quantitative analysis of proteins. The other three methods proved to be more suitable for the preparation of individual tear proteins but were less suitable for their quantitation.     

Separation of different physical forms of plasmid DNA using a combination of low electric field strength and flow in porous media: effect of different field gradients and porosity of the media

The retention of different physical forms of DNA by an electric field in a chromatography system was studied. We were able to effectively separate the supercoiled and the open circular forms of plasmid DNA using this type of electrochromatography system. Chromatography columns were packed with porous beads, and an axial electric field was applied so that convective buffer flow opposed the direction of electrophoresis of the DNA. A model system composed of approximately equal amounts of the super-coiled and open circular forms of the plasmid pBR 322 (4322 base pairs) was used to test the separation. Chromatography beads (agarose-based) with different porosities were used to determine the effect of the stationary phase on the separation. The porous media did not have a major effect on the separation, but the best separations were obtained using porous chromatography media made with the highest agarose concentration (10% agarose). Selective elution of plasmid DNA with different forms was obtained by either increasing the flow rates or decreasing the electric field strength (by steps or a gradient). In all the separations, the more compact supercoiled form of the plasmid was retained less strongly than either the open circular form (nicked) or the linear form. High molecular weight host genomic DNA was more strongly retained than the plasmid DNA. Increasing the ionic strength of the buffer improved resolution and capacity. The capacity of the separation was determined by injecting increasing amounts of plasmid DNA. Satisfactory separation was obtained at sample loading of up to 360 microg of total DNA on a column with dimensions of 2.5 by 11 cm (bed volume of 54 mL). The retention of DNA depends upon a counter-current flow of electrophoresis and convective flow and could be regarded as a type of field flow fractionation. The retention of the DNA by the electric field and flow is discussed in relation to the diffusion coefficients of the DNA.     

Construction of a Standard Reference Plasmid for Detecting GM Cottonseed Meal

A plasmid was constructed for quantification of genetically modified (GM) cottonseed meal in the gene-specific level. The Cry1Ab/c gene was connected with the Sad1 gene by fusion PCR. The fusion gene was cloned into the pMD®19-T Simple Vector. The plasmid DNA was then digested with a restriction endonuclease SmaI to reduce the characteristic differences between the plasmid DNA and genomic DNA. For a rough quantitative analysis of GM cotton meal contents, a rapid method for measurement of the copy numbers of the transgenic Cry and cotton endogenous Sad1 gene using a real-time PCR system with the plasmid DNA as a calibrator was established. The inter-run and intra-run coefficients of variation were less than 1.48% and 2.36%, respectively. The limits of detection and quantitation of the Cry and Sad1 genes were 9 and 91 copies of pMDCS, respectively. These results prove that the standard plasmid represents a valuable alternative to genomic DNA as a certified reference material for the quantification of GM cotton and is a useful tool to establish a feasible identification management for GM cottonseed meal content in the feed industry.     

Supercoiled plasmid quality assessment by analytical arginine-affinity chromatography

Supercoiled plasmids are an important component of gene-based delivery vehicles, applied in new therapeutic strategies such as gene therapy or DNA vaccination. However, aiming at the general distribution of plasmid DNA (pDNA) therapeutics requires a procedure to easily and efficiently assess the purity and recovery yield of the supercoiled (sc) plasmid isoform. Based on affinity interactions between amino acids and nucleic acids, an arginine affinity methodology with UV detection was established to quantify and to control the quality of sc plasmid biopharmaceuticals. The fact that this new technique allows to distinguish between plasmid isoforms represents an advantage, since it allows the selective quantification of the biologically active pDNA topology, and a more accurate analysis of the quality of the isolated plasmid. The analytical experiments were performed in 12 min and the method was found to be accurate, precise, reproducible and linear for a sc plasmid concentration range between 2 and 150 μg/mL. In comparison with other established methods used in the quantification of native pDNA (oc+sc), the main advance introduced by this new method is the possibility to quantify the sc plasmid in a sample containing other plasmid topologies, ensuring the purity of plasmid products to be therapeutically applied.     

质粒DNA在灌注色谱基质上的吸附行为

Anion exchange chromatography is the most popular chromatographic method for plasmid separation.POROS R1 50 is a perfusio n chro mato graphic suppo rt w hich is a reversed phase matrix and is an alternative to co nventio nal o nes due to its mass transfer pro perties.The adso rptio n and elutio n o f the pIDKE2 plasmid o nto reversed phase POROS R1 50 w as studied.Langmuir iso therm mo del w as adjusted in o rder to get the max imum adso rptio n capacity and the disso ciatio n co nstant fo r POROS R1 50-plasmid DNA(pDNA) system.Breakthro ugh curves w ere o btained fo r vo lumetric flo w s betw een 0.69-3.33 mL/min,given dynamic capacity up to 2.3 times higher than tho se repo rted fo r io nic ex change matrix used during the purificatio n pro cess o f plasmids w ith similar size to that o f pIDKE2.The efficiency w as less than 45% fo r the flo w co nditio ns and initial co ncentratio n studied,w hich means that the suppo rt w ill no t be o perated under saturatio n circumstances.     

Gene Sequence, Soluble Expression and Homologous Comparison of a D-Hydantoinase from Pseudomonas putida YZ-26

A 1440bp open-reading frame encoding D-hydantoinase from Pseudomonas putida YZ-26 was cloned and sequenced（ GenBank AY387829）. The DNA fragment was inserted into Nde and BamHI sites of vector pET-3a, yielding a recombinant plasmid, pET-HDT. After being transferred into the host strain, the artificial strain, pET-HDT/ E. coli BL21 can express the D-hydantoinase as the soluble form in the Lura-Bertani medium without addition of any inducers. The activity of the enzyme toward substrate DL-hydantoin can reach 3000-4000 IU per cells from one-liter bacterial culture incubated at 30 ℃ for 10-12 h. By the comparison of amino acid sequence homology, hydrophobic residues analysis and secondary structure prediction, it was found that D-hydantoinase reported herein is quite similar to that from Pseudomonas putdia CCRC12857, and alike to that from Pseudomonas putdia DSM84 or other bacteria. A rapid and efficient purification procedure of the enzyme was performed by a three-step procedure： ammonium sulfate fractionation, phenyl Sepharose hydrophobic interaction chromatography and Sephacryl S-200 gel filtration. The molecular mass of the monomeric enzyme is 52042 Da as determined by MALDI-TOF mass spectrometry.     

DNA与非离子糖基表面活性剂相互作用的研究

用动态表面张力法、键合等温线、紫外光谱及荧光光谱等方法研究了不同链长烷基葡萄糖苷(APG)与DNA的相互作用．研究发现APG对DNA键合可分为两阶段,第一阶段：多苷依靠多羟基结构与DNA形成动力学稳定的复合物;第二阶段：随时间延长,单苷由于其较小的空间位阻而与DNA形成能量更低的热力学稳定复合物．由平衡渗析法得到的单苷与DNA相互作用键合等温线显示,APG与DNA键合为一非协同过程．证实了其非离子氢键吸附的本质,同时也支持了DNA对胶束及预胶束的缠绕模型．紫外光谱结果证明了在APG与DNA作用过程中疏水作用的重要性．以溴化乙锭为探针,荧光光谱法研究证明,随APG链长增加,DNA构象缩拢程度加大,但即使是C2APG也仅能使DNA构象部分缩拢,推测DNA仅是部分链段对APG胶束进行包裹,其它链段仍处于伸展状态．与阳离子表面活性剂十六烷基三甲基溴化铵(CTAB)可使DNA构象强烈缩拢的事实相比,证明了静电作用在大分子与表面活性剂相互作用中的主导性．     

Adsorption of plasmid DNA to a natural organic matter-coated silica surface: kinetics, conformation, and reversibility

A quartz crystal microbalance with dissipation (QCM-D) has been used to determine the adsorption rate of ampicillin-resistant linear and supercoiled plasmid DNA onto a silica surface coated with natural organic matter (NOM). The structure of the resulting adsorbed DNA layer was determined by analyzing the viscoelastic properties of the adsorbed DNA layers as they formed and were then exposed to solutions of different ionic composition. The QCM-D data were complemented by dynamic light scattering measurements of diffusion coefficients of the DNA molecules as a function of solution ionic composition. The obtained results suggest that electrostatic interactions control the adsorption and structural changes of the adsorbed plasmid DNA on the NOM-coated silica surface. The adsorption of DNA molecules to the NOM layer took place at moderately high monovalent (sodium) electrolyte concentrations. A sharp decrease in solution ionic strength did not result in the release of the adsorbed DNA, indicating that DNA adsorption on the NOM-coated silica surface is irreversible under the studied solution conditions. However, the decrease in electrolyte concentration influenced the structure of the adsorbed layer, causing the adsorbed DNA to adopt a less compact conformation. The linear and supercoiled DNA had similar adsorption rates, but the linear DNA formed a thicker and less compact adsorbed layer than the supercoiled DNA.     

Electrophoretic behavior of plasmid DNA in the presence of various intercalating dyes

In the present study, the electrophoretic behavior of linear, supercoiled and nicked circular plasmid DNA in the presence of various intercalating dyes was characterized using pGL3 plasmid DNA as a model. The enzymatic digestion of pGL3 plasmid DNA with HindIIIwas monitored by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Nicked circular plasmid DNA was found to be relatively sensitive to enzymes, and was almost digested into the linear conformer after 10-min incubation, indicating that nicked circular plasmid DNA has little chance of targeting and entering the cell nucleus. Partly digested plasmid DNA containing only linear and supercoiled conformers can be used as a standard to confirm the migration order of plasmid DNA. In methylcellulose (MC) solution with YO-PRO-1 or YOYO-1, linear plasmid DNA eluted first, followed by supercoiled and nicked plasmid DNA, and nicked plasmid DNA eluted as a broad peak. With SYBR Green 1, nicked plasmid DNA eluted first as three sharp peaks, followed by linear and supercoiled plasmid DNA. The nuclear plasmid DNA from two transfected cell lines was successfully analyzed using the present procedure. Similar results were obtained with an analysis time of seconds using microchip electrophoresis with laser-induced fluorescence detection (mu-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or mu-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems.