Purification of plasmid DNA from Escherichia coli ferments using anion-exchange membrane and hydrophobic chromatography

A novel downstream bioprocess was developed to obtain purified plasmid DNA (pDNA) from Escherichia coli ferments. The intermediate recovery and purification of the pDNA in cell lysate was conducted using hollow-fiber tangential filtration and frontal anion-exchange membrane and elution hydrophobic chromatographies. The purity of the solutions of pDNA obtained during each process stage was investigated. The results show that the pDNA solution purity increased 30-fold and more than 99% of RNA in the lysate was removed during the process operations. The combination of membrane operations and hydrophobic interaction chromatography resulted in an efficient way to recover pDNA from cell lysates. A better understanding of membrane-based technology for the purification of pDNA from clarified E. coli lysate was developed in this research.     

Pilot-scale production and purification of a staphylokinase-based fusion protein over-expressed in Escherichia coli

SFH, a recombinant staphylokinase-based fusion protein linked by the factor Xa recognition peptide at the N-terminus of hirudin, is a promising therapeutic candidate for thromboembolic diseases. To develop SFH into a new thrombolytic agent, scaled-up production was carried out to provide sufficient preparation for animal safety and clinical studies. Here, we describe a pilot-scale cultivation and purification process for the production of SFH. A high-cell-density fed-batch cultivation for the production of SFH in E. coli was developed in a 40-L bioreactor, which produced about 1.1 g/L of recombinant protein. SFH was purified to homogeneity from the E. coli lysate by expanded bed adsorption chromatography and anion-exchange chromatography, with over 99% purity and 54% recovery. Moreover, the residual endotoxin content was less than 0.5 EU/mL. The molecular weight and in vitro bioactivity of SFH were also determined by electrospray ionization-mass spectrometry (ESI-MS) and fibrinolytic activity assay, respectively.     

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn²⁺ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.     

Flexprep: Scale-flexible rapid plasmid preparation for analysis of recombinant clones

A scale-flexible and cost-efective protocol for plasmid preparation is described to cover miniprep and midiprep scale work in a microcentriguge format for analysis of recombinant clones. this protocol relies on a modified alkaline lysis of Escherichia coli cells and subsequent purification of plasmid DNA with no organic extraction and alcohol precipitation. It can process up to 20 mL of E. coli cells carrying 3–10 kbp plasmid vectors in <10 min. Flexprep delivers sufficient yield and purity of plasmid DNA for routine applications including restriction enzyme digestion and fluorescent automated sequencing.     

Cytokine production using membrane adsorbers: Human basic fibroblast growth factor produced by Escherichia coli

Basic fibroblast growth factor (FGF‐2) is a multifunctional cytokine that regulates various cellular processes both in vitro and in vivo. FGF‐2 is extensively used in embryonic stem cell cultures since it can maintain the cells in an undifferentiated state. However, the high price of FGF‐2 has limited its application in stem cell research. Here we present a fast and efficient process for the purification of FGF‐2 from recombinant Escherichia coli cultures using reusable membrane adsorbers. A high expression level of FGF‐2 (42 mg/g dry cell) was achieved by fed‐batch cultivation of E. coli BL21(DE3). A new combination of cation exchange membrane chromatography and heparin‐sepharose affinity chromatography was used for the purification of the protein. A novel anion exchange membrane chromatography was used in the polishing step to remove endotoxins and DNA. In this new process, about 200 mg soluble FGF‐2 was yielded from 1.9 L culture broth with a purity of 98%. The purified protein was identified to be endotoxin‐free and bioactive. It was successfully tested to keep primate embryonic stem cell and human‐induced pluripotent stem cell pluripotent. Our approach, in which a controlled cultivation process is combined with an optimized fast and versatile downstreaming process, is suitable for low‐cost preparation of bioactive FGF‐2 at bench‐scale and may be beneficial to the effective production of other cytokines.     

Purification of plasmid DNA using a multicompartment electrolyser separated by ultrafilter membranes

A simple, scalable method for purification of plasmid DNA is described. Plasmid DNA was released from Escherichia coli JM109 by lysis (1% SDS, 0.2 M NaOH). Then a neutralization solution (3 M sodium acetate buffer, pH 4.8) was added to precipitate genomic DNA and protein. After the clarification of the lysate, the supernatant was placed in a multicompartment electrolyser separated by ultrafilter membranes to remove the remaining contamination (RNA, genomic DNA and protein). A recovery of 75%±2% of total plasmid DNA was obtained after 60 min electrophoresis with a field strength of 8 V cm^–1 using cells at 30 g l^–1 (quantified by dry cell weight). Genomic DNA, RNA and protein were undetectable in the purified plasmid DNA solution.     

Anion exchange purification of plasmid DNA using expanded bed adsorption

Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH=8.0) buffer at an upward flow of 300 cmh^-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh^-1. Purification factors of 36±1 fold, 26±0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed–values of 35±2 and 5±0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.     

DNA assay for recombinant pharmaceutical substances using the real-time PCR technique

A real-time PCR procedure is proposed for assaying E. coli residual DNA in the pharmaceutical substance of human recombinant insulin. For the quantitative analysis of the DNA content, an amplification of fragments of the bla gene plasmid DNA and E. coli genomic DNA of the 16S RNA gene were used. The contents of plasmid and genomic DNA were detected both in intermediates at various stages of the insulin purification process and in the finished product.     

Extraction of plasmid DNA using reactor scale alkaline lysis and selective precipitation for scalable transient transfection

DNA extracted and purified for vaccination, gene therapy or transfection of cultured cells has to meet different criteria. We describe herein, a scalable process for the primary extraction of plasmid DNA suitable for transient expression of recombinant protein. We focus on the scale up of alkaline lysis for the extraction of plasmid DNA from Escherichia coli, and use a simple stirred tank reactor system to achieve this. By adding a series of three precipitations (including a selective precipitation step with ammonium acetate) we enrich very quickly the plasmid DNA content in the extract. The process has been thus far used to extract up to 100 mg of plasmid from 1.5 l of clarified lysate, corresponding to an E.coli bioreactor fermentation of 3 l. This revised version was published online in July 2006 with corrections to the Cover Date.     

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。     

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD_260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.     

A colony‐to‐lawn method for efficient transformation of Escherichia coli

Aims: To develop a fast, convenient, inexpensive and efficient Escherichia coli transformation method for changing hosts of plasmids, which can also facilitate the selection of positive clones after DNA ligation and transformation. Methods and Results: A single fresh colony from plasmid‐containing donor strain is picked up and suspended in 75% ethanol. Cells are pelleted and resuspended in CaCl₂ solution and lysed by repetitive freeze–thaw cycles to obtain plasmid‐containing cell lysate. The E. coli recipient cells are scraped from the lawn of LB plate and directly suspended in the plasmid‐containing cell lysate for transformation. Additionally, a process based on colony‐to‐lawn transformation and protein expression was designed and conveniently used to screen positive clones after DNA ligation and transformation. Conclusions: With this method, a single colony from plasmid‐containing donor strain can be directly used to transform recipient cells scraped from lawn of LB plate. Additionally, in combination with this method, screening of positive clones after DNA ligation and transformation can be convenient and time‐saving. Significance and Impact of the Study: Compared with current methods, this procedure saves the steps of plasmid extraction and competent cell preparation. Therefore, the method should be highly valuable especially for high‐throughput changing hosts of plasmids during mutant library creation.     

Plasmid DNA production combining antibiotic‐free selection,inducible high yield fermentation,and novel autolytic purification

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage λR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non‐ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid–liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts. Biotechnol. Bioeng. 2009; 104: 505–515 © 2009 Wiley Periodicals, Inc.     

Isotopically labeled expression in E. coli, purification, and refolding of the full ectodomain of the influenza virus membrane fusion protein

This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of Escherichia coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A₆₀₀  8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the ¹³CO chemical shifts measured using solid-state nuclear magnetic resonance spectroscopy.     

重组质粒pVAX1-PENK大规模制备研究

目的：建立质粒pVAX1-PENK的大规模制备2--艺。方法：对大肠杆菌工程菌DH5α-pVAX1-PENK进行补料发酵,利用自行发明的连续碱裂解过程对菌体进行裂解,经超滤浓缩后,用Sepharnse 6 Fast Flow层析柱分离DNA与RNA,再经Plasmidselect Xtra层析柱分离超螺旋质粒DNA与开环或线性质粒DNA,最后经Source 15Q层析柱精制质粒DNA。结果：发酵获得质粒pVAX1-PENK的产率为182mg／L,经碱裂解及层析分离后,最终制备的质粒DNA超螺旋比例大于98％,总回收率为60.5％,纯度（D260nm／D280nm）为1.8～2.0。结论：建立的质粒DNA生产工艺可以制备大量高纯度的质粒DNA,并避免了使用动物源性的酶及有毒试剂。     

Downstream processing of high chain length polysialic acid using membrane adsorbers and clay minerals for application in tissue engineering

Polysialic acid (polySia) is a carbohydrate polymer of varying chain length. It is a promising scaffold material for tissue engineering. In this work, high chain length polySia was produced by an Escherichia coli K1 strain in a 10‐L bioreactor in batch and fed‐batch mode, respectively. A new downstream process for polySia is presented, based on membrane adsorber technology and use of inorganic anion exchanger. These methods enable the replacement of precipitation steps, such as acetone, cetavlon, and ethanol precipitation of the already established purification process. The purification process was simplified, while process efficiency and product qualities were improved. The overall yield of polySia from a 10‐L batch cultivation process was 61% and for 10‐L fed‐batch cultivation process the yield was 40% with an overall purity of 98%. The endotoxin content was determined to be negligible (14 EU mg^?1). The main advantage of this new downstream process is that polySia with high chain length of more than 130 degree of polymerization can be obtained. In fed‐batch cultivation, chain lengths up to 160 degree of polymerization were obtained.     

Optimization of continuous purification of recombinant patchoulol synthase from Escherichia coli with membrane adsorbers

The natural production of patchouli oil in developing countries cannot meet the increasing demand any more. This leads to socioecological consequences, such as the use of arable land, which is actually intended for food. Hence, the world market price increased up to $150/kg. An alternative is the biotechnological production of patchouli oil using a multiproduct sesquiterpene synthase, the patchoulol synthase (PTS). Here, we report the optimization of recombinant PTS purification from Escherichia coli lysate using continuous immobilized metal affinity chromatography. First, the purification conditions of the batch process were optimized in regard to optimal buffer composition and optimized chromatographic conditions. The best purification result was achieved with Co²⁺-immobilized metal affinity chromatography (Sartobind® IDA 75) with a triethanolamine buffer at pH 7, 0.5 M NaCl, 10% [vol/vol] glycerol, 5 mM MgCl₂ and 250 mM imidazole for product elution. This optimized method was then transferred to a continuous chromatography system using three membrane adsorber units (surface of 75 cm² each). Within 1.5 hr in total, 4.55 mg PTS with a final purity of 98% and recovery of 68% could be gained. The purified enzyme was used to produce 126 mg/L (-)-patchoulol from farnesyl pyrophosphate. Here, for the first time bioactive PTS was successfully purified using membrane adsorbers in a continuous downstream process.     

Combinatorial Method for Overexpression of Membrane Proteins in Escherichia coli

Membrane proteins constitute 20–30% of all proteins encoded by the genome of various organisms. Large amounts of purified proteins are required for activity and crystallization attempts. Thus, there is an unmet need for a heterologous membrane protein overexpression system for purification, crystallization, and activity determination. We developed a combinatorial method for overexpressing and purifying membrane proteins using Escherichia coli. This method utilizes short hydrophilic bacterial proteins, YaiN and YbeL, fused to the ends of the membrane proteins to serve as facilitating factors for expression and purification. Fourteen prokaryotic and mammalian membrane proteins were expressed using this system. Moderate to high expression was obtained for most proteins, and detergent solubilization combined with a short purification process produced stable, monodispersed membrane proteins. Five of the mammalian membrane proteins, overexpressed using our system, were reconstituted into liposomes and exhibited transport activity comparable with the native transporters.     

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.     

Time-course determination of plasmid content in eukaryotic and prokaryotic cells using Real-Time PCR

A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1 derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires neither specific cell lysis nor DNA purification and can be performed in <30 min with dynamic ranges covering 0.9 pg–55 ng, and 5.0 pg–2.5 ng of plasmid DNA (pDNA) for E. coli and CHO cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h post-transfection was around 3 × 10⁴ copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and around 100 copies/cell were still detected 6 days after transfection.