Genetic analysis of a lactococcal plasmid replicon

Summary The sequence and genetic organization was determined of the 2508 by lactococcal portion of pFX2, which was derived from a crypticLactococcus lactis subsp.lactis plasmid and used as the basis for construction of a series of lactococcal vectors. A lactococcal plasmid plus origin and two replication protein-coding regions (repA andrepB) were located. RepA has a helix-turn-helix motif, a geometry typical of DNA-binding proteins. RepB shows a high degree of homology to the plasmid replication initiation proteins from other gram-positive bacteria andMycoplasma. The transcribed inverted repeat sequence betweenrepA andrepB could form an attenuator to regulate pFX2 replication. Upstream of theori site, and in a region which was non-essential for replication, a 215 by sequence identical to the staphylococcal plasmid pE194 and carrying the RS_A site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with pE194 group plasmids.     

The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells

Epstein-Barr virus (EBV) is an exceptionally successful human viral pathogen maintained as a licensed, plasmid replicon in proliferating cells. We have measured the distributions of EBV-derived plasmids in single live cells throughout the cell cycle in the absence of selection and confirmed the measured rates of duplication and partitioning computationally and experimentally. These analyses have uncovered a striking, non-random partitioning for this minimalist plasmid replicon and revealed additional properties of it and its host cells: (1) 84% of the plasmids duplicate during each S phase; (2) all duplicated plasmids are spatially colocalized as pairs, a positioning that is coupled to their non-random partitioning; (3) each clone of cells requires a certain threshold number of plasmids per cell for its optimal growth under selection; (4) defects in plasmid synthesis and partitioning are balanced to yield wide distributions of plasmids in clonal populations of cells for which the plasmids provide a selective advantage. These properties of its plasmid replicon underlie EBV's success as a human pathogen.     

大规模生产制备质粒DNA技术进展

质粒DNA疫苗或基因治疗剂是生物制品的新品种。鉴于质粒DNA表达效率低、持续时间短,需建立一套大规模生产制备质粒DNA的工艺,即发酵、碱性裂解、分离纯化及质量控制。目前质粒DNA大规模生产已经达到千克级水平,一些产品正在进行临床实验。     

A procedure for large-scale isolation of RNA-free plasmid and phage DNA without the use of RNase

A preparative procedure for the large-scale isolation of plasmid DNA without the use of RNAse is described. Crude plasmid DNA is prepared using a standard boiling method. High-molecular-weight RNA is removed by precipitation with LiCl, and low-molecular-weight RNA is removed by sedimentation through high-salt solution. The procedure is inexpensive, rapid, simple, and particularly suitable for processing several large-scale preparations simultaneously. A similar procedure has been developed for preparation of lambda-phage DNA.     

Alkaline-cell lysis through in-line static mixer reactor for the production of plasmid DNA for gene therapy

A state-of-the-art in-line static mixer reactor (ISMR) was invented to lyse E. coli cells and neutralize the cell lysate continuously and efficiently for the extraction of plasmid DNA. It comprised two connected static dynamic mixers, each 0.01 m in diameter and 0.9 m in length, one for lysis and one for neutralization. Cells were lysed using concentrated alkaline with 1% SDS and the lysate was neutralized at feed rates of cell suspension:lysis solution:neutralization solution of 125:250:125, 250:500:250, and 500:1,000:500 mL/min. Distances for the mixtures to reach color homogeneity were dependent on feed rates. The higher the feed rates the shorter the mixing distances and times. However, complete cell lysis and neutralization were independent of color homogeneity. Lysate viscosity and neutralized floc size decreased and floc density increased, as distances and feed rates increased. High plasmid yields were obtained from both lysis and neutralization at feed rate ratios of 125:250:125 and 250:500:250 mL/min within mixing distances < or =0.6 m. Poor mixing performance and plasmid yield were obtained at a high feed rate of 500:1,000: 500 mL/min when residence and reaction times were less than 2 s and from mixing distances > or =0.6 m at all feed rates due to a longer exposure to strong alkali and shear flow. This invention showed excellent performance with scaleable potential for the commercial manufacture of plasmid DNA.     

A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy

With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase‐mediated plasmid supercoiling. SGS from three different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57‐SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total percentage supercoiled monomer was maintained to 85–90%. A twofold increase in plasmid yield was also observed for pUC57‐SGS in comparison to pUC57. pUC57‐SGS displayed greater segregational stability than pUC57‐cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. Biotechnol. Bioeng. 2016;113: 2064–2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large-scale processes

We set out to investigate if E. coli genotype plays a significant role in host strain selection for optimal processing of plasmid DNA based on both quality and quantity of supercoiling. Firstly 17 E. coli commercial and non-commercial strains were selected and their available genetic backgrounds were researched in the open literature. Growth characteristics of all the strains were considered and made impartial by using a common medium and growth condition platform. By keeping the growth conditions constant for each strain/plasmid combination, we are only looking at one variable which is the host strain. The second step was to attempt to correlate the findings with common genotype characteristics (e.g. mutations such as endA or recA). We found that one can screen the number of strains which are likely to give good productivity early on, before any further optimisation and verification is performed, both for small and large plasmids. Also, it is worth noting that complex plasmid interactions with each strain prevent the use of genotype alone in making an intelligent choice for supercoiling optimisation. This leads to a third optimisation step selecting a few of the potentially high performing strains based on high DNA yield and supercoiling, with a view to identify the factors which need improvement in strain design and bioreactor optimisation. We found that high specific growth rates of some strains did not affect the level of DNA supercoiling but did influence the total plasmid yield, potentially an important aspect in the design of fermentation strategy. Interestingly, a few host/plasmid combinations result in what appears to be runaway plasmid replication.     

Separation of genomic DNA, RNA, and open circular plasmid DNA from supercoiled plasmid DNA by combining denaturation, selective renaturation and aqueous two-phase extraction

In the current study we developed a process for the capture of pDNA exploiting the ability of aqueous two-phase systems to differentiate between different forms of DNA. In these systems scpDNA exhibits a near quantitative partitioning in the salt-rich bottom phase. The successive recovery from the salt rich bottom phase is accomplished by a novel membrane step. The polish operation to meet final purity demands is again based on a system exploiting a combination of the denaturation of the nucleic acids present, specific renaturation of scpDNA, and an ATP system able to differentiate between the renatured scpDNA and the denatured contaminants such as ocpDNA and genomic host DNA. This polish step thus allows a rapid and efficient separation of scpDNA from contaminating nucleic acids which up to date otherwise only can be accomplished with much more cumbersome chromatographic methods. In a benchmark comparison, it could be shown that the newly developed process exhibits a comparable yield to an industrial standard process while at the same time showing superior performance in terms of purity and process time. Additionally it could be shown that the developed polish procedure can be applied as a standalone module to support already existing processes.     

Fractional precipitation of plasmid DNA from lysate by CTAB

Preparative-scale purification of plasmid DNA has been attempted by diverse methods, including precipitation with solvents, salts, and detergents and chromatography with ion-exchange, reversed-phase, and size-exclusion columns. Chromatographic methods such as hydrophobic interaction chromatography (HIC), reversed phase chromatography (RPC), and size exclusion chromatography (SEC) are the only effective means of eliminating the closely related relaxed and denatured forms of plasmid as well as endotoxin to acceptable levels. However, the anticipated costs of manufacturing-scale chromatography are high due to (a) large projected volumes of the high-dosage therapeutic molecule and (b) restricted loading of the large plasmid molecule in the pores of expensive resins. As an alternative to chromatography, we show herein that precipitation with the cationic detergent, cetyltrimethylammonium bromide (CTAB), is effective for selective precipitation of plasmid DNA from proteins, RNA, and endotoxin. Moreover, CTAB affords novel selectivity by removal of host genomic DNA and even the more closely related relaxed and denatured forms of plasmid as earlier, separate fractions. Finally, plasmid that has been precipitated by CTAB can be purified by selectively dissolving under conditions of controlled salt concentration. The selectivity mechanism is most likely based upon conformational differences among the several forms of DNA. As such, CTAB precipitation provides an ideal nonchromatographic capture step for the manufacture of plasmid DNA.     

Cloning and characterization of a replicon region of the IncHII plasmid pHH1457

A replicative region of the large conjugative plasmid pHH1457 (incompatibility group HII (IncHII)) was cloned. A 1.4-kbp region, in a stable pSBII14 clone, containing a PolI-independent replicon and determinants for the HII incompatibility phenotype, was selected and characterized. High incompatibility with IncHII plasmids was corroborated. Independent replication of the insert was demonstrated by ligation to an antibiotic resistance cassette. pSBII14 was used as a probe to identify IncHII plasmids from other members of the H complex: IncHI (IncHI1, IncHI2 and IncHI3 subgroups). Hybridization experiments revealed a high homology with the replication region of IncHII plasmids, but not with IncHI1 or IncHI3 plasmid prototypes. Homology with IncHI2 plasmids was observed, suggesting the presence of IncHII-like replicons among this subgroup of plasmids. This is the first report of the characterization of an IncHII plasmid maintenance region.     

Rapid plasmid DNA isolation from Lactococcus lactis using overnight cultures

A simple and quick method has been developed to isolate plasmid DNA from Lactococcus lactis using overnight or stationary-phase cultures which therefore eliminates the need for subculturing for generating log-phase cultures that are necessary with existing methods. The new method was effective in isolating plasmids, from 1.4 to 64 kb, from the three subspecies of Lactococcus lactis. The resultant DNA was of high yield and purity and therefore no additional purification steps were required for down-stream molecular procedures.     

阴离子交换晶胶层析分离质粒DNA

质粒DNA(pDNA)作为重要的基因治疗药物载体,其广泛应用受纯度和产量的限制。为了获得高纯度的pDNA,首先制备超大孔连续床晶胶基质,接枝二乙氨基乙基葡聚糖得到阴离子交换型晶胶介质;然后以pUC19质粒为例,将目标质粒转化至大肠杆菌,培养收集,碱液裂解和离心;最后用阴离子交换型晶胶介质从离心上清液中一步法层析分离pDNA。通过优化层析过程的pH值和洗脱条件,最终在pH值为6.6时,用0.5 mol/L的NaCl溶液洗脱,得到较高纯度的pDNA。整个分离过程中不使用动物源性酶,也不需常规分离中的高毒试剂,使获得pDNA的过程和产物更加安全。     

Fed-batch microbioreactor platform for scale down and analysis of a plasmid DNA production process

The rising costs of bioprocess research and development emphasize the need for high-throughput, low-cost alternatives to bench-scale bioreactors for process development. In particular, there is a need for platforms that can go beyond simple batch growth of the organism of interest to include more advanced monitoring, control, and operation schemes such as fed-batch or continuous. We have developed a 1-mL microbioreactor capable of monitoring and control of dissolved oxygen, pH, and temperature. Optical density can also be measured online for continuous monitoring of cell growth. To test our microbioreactor platform, we used production of a plasmid DNA vaccine vector (pVAX1-GFP) in Escherichia coli via a fed-batch temperature-inducible process as a model system. We demonstrated that our platform can accurately predict growth, glycerol and acetate concentrations, as well as plasmid copy number and quality obtained in a bench-scale bioreactor. The predictive abilities of the micro-scale system were robust over a range of feed rates as long as key process parameters, such as dissolved oxygen, were kept constant across scales. We have highlighted plasmid DNA production as a potential application for our microbioreactor, but the device has broad utility for microbial process development in other industries as well.     

Degradation of supercoiled plasmid DNA within a capillary device

Supercoiled plasmid DNA is susceptible to fluid stress in large-scale manufacturing processes. A capillary device was used to generate controlled shear conditions and the effects of different stresses on plasmid DNA structure were investigated. Computational fluid dynamics (CFD) analysis was employed to characterize the flow environment in the capillary device and different analytical techniques were used to quantify the DNA breakage. It was found that the degradation of plasmid DNA occurred at the entrance of the capillary and that the shear stress within the capillary did not affect the DNA structure. The degradation rate of plasmids was well correlated with the average elongational strain rate or the pressure drop at the entrance region. The conclusion may also be drawn that laminar shear stress does not play a significant role in plasmid DNA degradation.     

A DNA replicon system for rapid high‐level production of virus‐like particles in plants

Recombinant virus‐like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low‐level antigen accumulation and long‐time frame to produce transgenic plants are the two major roadblocks in the practical development of plant‐based VLP production. In this article, we describe the optimization of geminivirus‐derived DNA replicon vectors for rapid, high‐yield plant‐based production of VLPs. Co‐delivery of bean yellow dwarf virus (BeYDV)‐derived vector and Rep/RepA‐supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co‐expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built‐in Rep/RepA cassette without P19 drove protein expression at similar levels as the three‐component system. These results demonstrate the advantages of fast and high‐level production of VLP‐based vaccines using the BeYDV‐derived DNA replicon system for transient expression in plants. Biotechnol. Bioeng. 2009;103: 706–714. © 2009 Wiley Periodicals, Inc.     

Separation of supercoiled from open circular forms of plasmid DNA,and biological activity detection

To establish a cost-effective purification process for the large-scale production of plasmid DNA for gene therapy and DNA vaccination, a single anion-exchange chromatography (AEC) step was employed to purify supercoiled plasmid DNA (sc pDNA) from other isoforms and Escherichia coli impurities present in a clarified lysate. Two different size and conformation plasmids were used as model targets, and showed similar elution behavior in this chromatographic operation, in which sc pDNA was effectively separated from open circle plasmid DNA (oc pDNA) in a salt gradient. The process delivered high-purity pDNA of homogeneity of 95 ± 1.1% and almost undetectable levels of endotoxins, genomic DNA, RNA and protein, at a yield of 65 ± 8%. Furthermore, the transfection efficiency (29 ± 0.4%) was significantly higher than that (20 ± 0.1%) of a pDNA control. The present study confirms the possibility of using a single AEC step to purify sc pDNA from other isoforms and host contaminants present in a clarified E. coli lysate.     

Genetic analysis of a novel plasmid pZMX101 from Halorubrum saccharovorum: determination of the minimal replicon and comparison with the related haloarchaeal plasmid pSCM201

The DNA sequence of a novel haloarchaeal plasmid pZMX101 (3918 bp) from Halorubrum saccharovorum was determined and six ORFs were predicted. The largest ORF encodes a putative replication initiation protein RepA, which shares 40% sequence similarity with the Rep201 of a theta-replication plasmid pSCM201 recently isolated from Haloarcula, suggesting that pZMX101 might replicate via a theta-type mechanism. Using pZMX101 as the only haloarchaeal replicon, a shuttle vector pZMX108 was constructed and successfully transformed into Haloferax volcanii DS70. Based on this in vivo system, the minimal replicon (1978 bp) of pZMX101 was determined. It is composed of the repA gene plus c. 400-bp upstream and 300-bp downstream sequences. Significantly, the putative replication origin of pZMX101 and that of pSCM201 contain different types of sequence motifs, and these two plasmids exhibit distinct host preference for Haloferax and Haloarcula, respectively.     

Amphiphilic peptides with arginines and valines for the delivery of plasmid DNA

A non-toxic and efficient gene carrier is one requirement for clinical gene therapy. In this study, amphiphilic peptides composed of arginines and valines were synthesized and characterized as plasmid DNA (pDNA) carriers. The peptides have a cationic region containing 1-4 arginines and a hydrophobic region containing 6 valines. The arginine-valine peptides (RV peptides) formed micelles in aqueous solution with a critical micelle concentration (CMC) of 1.35 mg/ml. In gel retardation assay, the RV peptides retarded all pDNA at weight ratios (pDNA:RV peptide) of 1:3 for R1V6, 1:2 for R2V6 and R3V6, and 1:1 for R4V6. A heparin competition assay showed that the R3V6 peptide formed tighter complexes with pDNA than poly-L-lysine (PLL). In vitro transfection assay into HEK293 cells showed that the R1V6 and R2V6 peptides had the highest transfection efficiencies at 1:30 weight ratios (pDNA:RV peptide), while the R3V6 and R4V6 peptides had the highest efficiencies at 1:20 weight ratios. Under optimal conditions, the R3V6 peptide had the highest transfection efficiency of all the RV peptides and PLL. MTT assay showed that the RV peptides did not have any detectable toxicity to cells. Therefore, the RV peptide may be useful for the development of non-toxic gene carriers.