Preparative separation of proteins using centrifugal precipitation chromatography based on solubility in ammonium sulfate solution

A preparative modification of the centrifugal precipitation chromatography (CPC) is described. The sample-loading capacity is improved in the present system by the use of convoluted tubing containing dialysis tubing instead of a dialysis membrane placed between a pair of disks equipped with mirror-imaged spiral grooves as in the original design. The system uses, basically, the same principle of as the original CPC, in that a concentration gradient of precipitant is generated under a centrifugal force field. The protein sample injected into the CPC column is exposed to an increasing concentration of the precipitant where it precipitates at various portions of the column according to its solubility. The gradient is then gradually lowered so that the sample undergoes dissolution and precipitation many times within the column; the proteins finally elute from the column according to their solubilities. A basic study was performed using this machine to separate human albumin and 3-globulin using ammonium sulfate (AS) as precipitant. Preliminary results indicate that this method can separate 500 mg of protein.     

Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH(4)Cl

Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel and solutes of interest undergo repetitive precipitation-dissolution, fractionate at different locations, and elute out from the channel according to their solubility in the precipitating agent solution. We report here for the first time the use of a CPC system for fractionation of protein, RNA, and plasmid DNA in clarified lysate produced from bacterial culture. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was initially used as a precipitating agent; however, all biomolecules showed no differential solubility in the moving concentration gradient of this surfactant and, as a result, no separation of protein, RNA, and plasmid DNA occurred. To overcome this problem, inorganic salts such as NaCl and NH(4)Cl were introduced into solution of CTAB. The protein and RNA were found to have higher solubility with the addition of these salts and separated from the plasmid DNA. Decreasing surface charge density of CTAB upon addition of NaCl and NH(4)Cl was believed to lead to lower surfactant complexation, and therefore caused differential solubility and fractionation of these biomolecules. Addition of CaCl(2) did not improve solubility and separation of RNA from plasmid DNA.     

Purification of plasmid DNA using tangential flow filtration and tandem anion-exchange membrane chromatography

A new bioprocess using mainly membrane operations to obtain purified plasmid DNA from Escherechia coli ferments was developed. The intermediate recovery and purification of the plasmid DNA in cell lysate was conducted using hollow-fiber tangential filtration and tandem anion-exchange membrane chromatography. The purity of the solutions of plasmid DNA obtained during each process stage was investigated. The results show that more than 97% of RNA in the lysate was removed during the process operations and that the plasmid DNA solution purity increased 28-fold. One of the main characteristics of the developed process is to avoid the use of large quantities of precipitating agents such as salts or alcohols. A better understanding of membrane-based technology for the purification of plasmid DNA from clarified E. coli lysate was developed in this research. The convenience of anion-exchange membranes, configured in ready-to-use devices can further simplify large-scale plasmid purification strategies.     

Separation of plasmid DNA from protein and bacterial lipopolysaccharides using displacement chromatography

The preparation of plasmid DNA at large scale constitutes a pressing problem in bioseparation. This paper describes a first investigation of displacement chromatography as a means to separate plasmid DNA (4.7 kb) from E. coli lipopolysaccharides and protein (holo transferrin), respectively. Displacement chromatography has advantages in this regard, since the substance mixture is resolved into rectangular zones of the individual components rather than into peaks. Thus a higher total concentration can be maintained in the pooled product fractions. Hydroxyapatite (type I and II) and anion exchange stationary phases were included in the experiments. In addition to a conventional anion exchange column packed with porous particles, the recently introduced continuous bed UNOTM anion exchange column was investigated. No DNA purification was possible with either hydroxyapatite material. Conventional particle based columns in general were not suited to the separation of any two substances varying considerably in molecular mass, e.g. plasmid DNA and standard protein. Presumably, the direct competition for the binding sites, which is essential in displacement chromatography, was restricted by the size dependency of the accessible stationary phase surface area in this case. Better results were obtained with the continuous bed column, in which the adsorptive surface coincides with the walls of the flow through pores. As a result the accessible surface does not vary as much with the size of the interacting molecules as for the conventional stationary phase materials. Sharper transitions were also observed between substance zones recovered from the UNOTM column. The steric mass action model was used to aid method development in case of the anion exchange approach. While further research in obviously necessary, displacement chromatography on continuous bed columns has been shown to be capable of separating plasmid DNA from typical impurities. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Centrifugal precipitation chromatography: principle, apparatus, and optimization of key parameters for protein fractionation by ammonium sulfate precipitation

A novel chromatographic system introduced here internally generates a concentration gradient of ammonium sulfate (AS) through a long separation channel under a centrifugal force field. Protein samples are exposed to a gradually increasing AS concentration and precipitated along the channel. Then, chromatographic elution is initiated by gradually decreasing the AS concentration in the gradient which causes the proteins to repeat dissolution and precipitation through the channel. Consequently, they are eluted out in the order of their solubility in the AS solution. The separation column consists of a pair of disks equipped with mutually mirror-imaged spiral grooves. A dialysis membrane is sandwiched between the disks to form two identical channels partitioned by the membrane. The disk assembly is mounted on the sealless continuous-flow centrifuge. When a concentrated AS solution is eluted through one channel and water through the other channel in an opposite direction, an exponential AS gradient is formed through the water channel. A series of basic experiments was performed to study the rates of AS transfer and osmosis through the membrane, and the operational parameters including elution time, revolution speed, inclination of gradient, and sample size were optimized using stable protein samples. Preliminary applications were successful in purification of monoclonal antibody from cell culture supernatant and an affinity separation of recombinant ketosteroid isomerase from a crude Escherichia coli lysate.     

Membrane protein identifications by mass spectrometry using electrocapture-based separation as part of a two-dimensional fractionation system

A two-dimensional (2D) separation method was used to decrease sample complexity in analysis of tryptic peptides from glomerular membrane proteins by tandem mass spectrometry (MS/MS). The first dimension was carried out by electrocapture (EC), which fractionates peptides according to electrophoretic mobility. The second dimension was reverse-phase liquid chromatography (RP-LC), in which EC fractions were further separated and analyzed online by MS/MS. Using this methodology, we now identify 102 glomerular proteins (57 membrane proteins). Many peptides were possible to observe and select for MS/MS only using the 2D approach. Others were detectable in both one-dimensional (1D, without the EC step) and 2D experiments but were selectable for sequence analysis only from the 2D separations because the decrease in complexity then gives time for the mass analyzer to select the peptide and switch to the MS/MS mode. A minority of the peptides were detectable only in the 1D mode (presumably because of handling losses), but at the end this did not decrease the number of proteins identified by the 2D separation. After a database search, the combination of EC and RP-LC MS/MS versus a 1D RP-LC MS/MS separation resulted in a threefold increase in the number of proteins identified and improved the sequence coverage in the identifications, bringing our proteome-identified glomerular proteins to 282.     

Large-scale purification of plasmid DNA by fast protein liquid chromatography using a Hi-Load Q Sepharose column.

The large-scale purification of plasmid DNA was achieved using fast protein liquid chromatography on a Hi-Load Q Sepharose column. This method allows for the purification of plasmids starting from crude plasmid DNA, prepared by a simple alkaline lysis procedure, to pure DNA in less than 5 h. In contrast to the previously described plasmid purification methods of CsCl gradient centrifugation or high-pressure liquid chromatography, this method does not require the use of any hazardous or expensive chemicals. More than 100 plasmids varying in size from 3 to 15 kb have been purified using this procedure. A Mono Q Sepharose column was initially used to purify plasmids smaller than 8.0 kb; however, a Hi-Load Q Sepharose column proved more effective with plasmids larger than 8 kb. The loading of plasmids larger than 8 kb on the Mono Q column resulted in a high back pressure and the plasmid DNA could not be eluted from the column. Thus, for routine purification we utilize the Hi-Load Q Sepharose column. Plasmids purified by this method had purity, yield, and transfection efficiency in mammalian cells similar to those of plasmids purified by CsCl density gradient centrifugation.     

Purification of single-strand DNA binding protein from an Escherichia coli lysate using counter-current chromatography,partition and precipitation

Single-strand DNA binding protein (SSB) from Escherichia coli lysate was purified by counter-current chromatography (CCC) using the ammonium sulfate precipitation method in a coiled column. About 5 ml of E. coli lysate was separated by CCC using a polymer phase system composed of 16% (w/w) polyethylene glycol (PEG) 1000 and 17% (w/w) ammonium sulfate aqueous polymer two-phase solvent system. The precipitation of proteins in the lysate took place in the CCC column, and the SSB protein was eluted in the fraction 51-56. Many other impurities were either eluted immediately after the solvent front or precipitated in the column. The identities of the proteins in the fractions and in the precipitate were confirmed by SDS-polyacrylamide gel electrophoresis with Coomassie Brilliant Blue staining.     

Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography

Hydroxyapatite and related stationary phases increasingly play a role in the downstream processing of high-value biological materials, such as recombinant proteins, therapeutic antibodies and pharmaceutical-grade plasmid DNA. Chromatographic hydroxyapatite is an inorganic, ceramic material identical in composition, if not in structure, to calcium phosphate found in human bones and teeth. The interaction of hydroxyapatite with biomacromolecules is complex and highly dynamic, which can make predicting performance difficult, but also allows the design of very selective isolation processes. This review discusses the currently commercially available chromatographic materials, different retention mechanisms supported by these materials and differential exploitation for the design of highly specific isolation procedures. The state of the art of antibody purification by hydroxy- and fluoroapatite is reviewed together with tested routines for method development and implementation. Finally, the isolation of plasmid DNA is discussed, since the purification of DNA therapeutics at a sufficiently large scale is an emerging need in bioprocess development and perhaps the area in bioseparation where apatite chromatography can make its most important contribution to date.     

Milligram scale parallel purification of plasmid DNA using anion-exchange membrane capsules and a multi-channel peristaltic pump

A parallel chromatographic procedure for the purification of milligram amounts of plasmid DNA was developed. Initial studies showed that ion-exchange membrane capsules displayed high capacity for plasmid DNA. Interestingly, a weak anion exchanger (DEAE) proved to be superior to the strong quarternary ammonium group with respect to elution and regeneration properties and the 75 cm(2) Sartobind D membrane capsule (MA75D, Sartorius) was selected for further studies. A method for reducing endotoxin levels by using CTAB as a precipitant was optimised. By introducing this step into the protocol, endotoxin levels could be reduced approximately 100-fold to 相似文献   

Secretion of a peripheral membrane protein, MFG-E8, as a complex with membrane vesicles.

MFG-E8 (milk fat globule-EGF factor 8) is a peripheral membrane glycoprotein, which is expressed abundantly in lactating mammary glands and is secreted in association with fat globules. This protein consists of two-repeated EGF-like domains, a mucin-like domain and two-repeated discoidin-like domains (C-domains), and contains an integrin-binding motif (RGD sequence) in the EGF-like domain. To clarify the role of each domain on the peripheral association with the cell membrane, several domain-deletion mutants of MFG-E8 were expressed in COS-7 cells. The immunofluorescent staining of intracellular and cell-surface proteins and biochemical analyses of cell-surface-biotinylated and secreted proteins demonstrated that both of the two C-domains were required for the membrane association. During the course of these studies for domain functions, MFG-E8, but not C-domain deletion mutants, was shown to be secreted as membrane vesicle complexes. By size-exclusion chromatography and ultracentrifugation analyses, the complexes were characterized to have a high-molecular mass, low density and higher sedimentation velocity and to be detergent-sensitive. Not only such a exogenously expressed MFG-E8 but also that endogenously expressed in a mammary epithelial cell line, COMMA-1D, was secreted as the membrane vesicle-like complex. Scanning electron microscopic analyses revealed that MFG-E8 was secreted into the culture medium in association with small membrane vesicles with a size from 100 to 200 nm in diameter. Furthermore, the expression of MFG-E8 increased the number of these membrane vesicle secreted into the culture medium. These results suggest a possible role of MFG-E8 in the membrane vesicle secretion, such as budding or shedding of plasma membrane (microvesicles) and exocytosis of endocytic multivesicular bodies (exosomes).     

Construction of an expression plasmid (vector) encoding Brucella melitensis outer membrane protein,a candidate for DNA vaccine

Background:

DNA vaccination with plasmid encoding bacterial, viral, and parasitic immunogens has been shown to be an attractive method to induce efficient immune responses. Bacteria of the genus Brucella are facultative intracellular pathogens for which new and efficient vaccines are needed.

Methods:

To evaluate the use of a DNA immunization strategy for protection against brucellosis, a plasmid containing the DNA encoding the Brucella melitensis (B. melitensis) 31 kDa outer membrane protein, as a potent immunogenic target, was constructed.

Results:

The constructed plasmid, pcDNA3.1+omp31, was injected intramuscularly into mice and the expression of omp31 RNA was assessed by RT-PCR. The integrity of the pcDNA3.1+omp31 construct was confirmed with restriction analysis and sequencing. Omp31 mRNA expression was verified by RT-PCR.

Conclusion:

Our results indicate that the pcDNA3.1+omp31 eukaryotic expression vector expresses omp31 mRNA and could be useful as a vaccine candidate.Key Words: Brucella melitensis, omp31, DNA Vaccine, pcDNA3.1     

Homologous human blood protein separation using immobilized metal affinity chromatography: protein C separation from prothrombin with application to the separation of factor IX and prothrombin.

Protein C (PC) is a natural anticoagulant and antithrombotic present in human blood at a concentration of 4 microg/mL. Its deficiency can result in excessive clotting and thrombosis. Protein C can be obtained from human blood plasma; however, there are other coagulant proteins in blood, including prothrombin (factor II), which is present in relatively large amounts and is one of the most active components. Protein C and prothrombin are homologous proteins with similar biochemical features; therefore, immunoaffinity chromatography is used for their separation. However, this technology is very expensive, protein C recovery and activity is low, and contamination problems with mouse antibody are likely. Immobilized metal affinity chromatography (IMAC) utilizes the protein metal-binding properties for protein separation. Protein C has twelve surface-accessible histidines, which are the major metal-binding groups for IMAC separation. After investigating metal ion-binding properties of protein C, we used an IDA-Cu column to separate protein C and prothrombin. Following protein adsorption to the column, prothrombin was washed out using a sodium phosphate buffer containing 2 mM imidazole and protein C was recovered with 15 mM imidazole in the buffer. The mild elution condition allows a high protein C activity and a high recovery. Also, this technology introduces no immunoglobulins, and it is relatively inexpensive. IMAC could replace the immunoaffinity technology for the large-scale separation of protein C from blood plasma Cohn Fraction IV-1. In addition, this work demonstrates a significant application of this technology for the separation of factor IX from prothrombin. Prothrombin has proven to be a harmful contaminant in factor IX cocktails that have been administered to humans in the treatment of hemophilia B.     

Biogenesis of a putative channel protein, ComEC, required for DNA uptake: membrane topology, oligomerization and formation of disulphide bonds

ComEC is a putative channel protein for DNA uptake in Bacillus subtilis and other genetically transformable bacteria. Membrane topology studies suggest a model of ComEC as a multispanning membrane protein with seven transmembrane segments (TMSs), and possibly with one laterally inserted amphipathic helix. We show that ComEC contains an intramolecular disulphide bond in its N-terminal extracellular loop (between the residues C131 and C172), which is required for the stability of the protein, and is probably introduced by BdbDC, a pair of competence-induced oxidoreductase proteins. By in vitro cross-linking using native cysteine residues we show that ComEC forms an oligomer. The oligomerization surface includes a transmembrane segment, TMS-G, near the cytoplasmic C-terminus of ComEC.     

Automated on-like dialysis, trace enrichment and high-performance liquid chromatography Inhibition of interaction with the dialysis membrane and disruption of protein binding in the determination of clozapine in human plasma

Problems related to interaction of drugs with the dialysis membrane and to protein binding must be overcome in order to develop automated methods for drug analysis based on on-line dialysis, trace enrichment and HPLC. In order to study these problems, clozapine and its active metabolite N-desmethylclozapine were chosen as model compounds because they were found to interact with the dialysis membrane, and clozapine is highly protein bound. Addition of a cationic surfactant, dodecylethyldimethyl ammonium bromide, to the donor solution and to the plasma samples was found to inhibit interaction of the drugs with surfaces. The protein binding in plasma was disrupted prior to dialysis by lowering the pH with hydrochloric acid and the plasma proteins were solubilised with glycerol. The results obtained were used to develop a fully automated method for the determination of clozapine and N-desmethylclozapine in human plasma. More than 100 samples could be analysed within 24 h. The limit of detection in human plasma was 0.050 μmol/1 for clozapine and 0.055 μmol/1 for N-desmethylclozapine. Linearity was found for drug concentrations between 0.25–3 μmol/1. The relative standard deviations were between 1.2–6.7% and the method was applicable for therapeutic drug monitoring.     

Quantitative determination of thalidomide in human serum with high-performance liquid chromatography using protein precipitation with trichloroacetic acid and ultraviolet detection

A validated and precise reversed-phase high-performance liquid chromatographic method for the determination of thalidomide in serum, with phenacetin as an internal standard, is described. Protein precipitation, using trichloroacetic acid, was used for clean-up. The aliquot was chromatographed on a octadecyl column, using an eluent composed of 250 ml 0.01 M potassium dihydrogenphosphate, adjusted to a pH of 3.0 with a 43% phosphoric acid solution, mixed with 750 ml methanol. Ultraviolet detection was used at an operation wavelength of 220 nm. Hydrolytic degradation was prevented during analysis by acidification of samples with the precipitation reagent. Thalidomide and phenacetin were found to have retention times of 7.9 and 15.0 min, respectively. Recoveries ranging from 79 to 84% were found for both components, with reproducibility relative standard deviations of 0.8–3% and repeatability coefficients of 1.2–3%. A mean correlation coefficient of 0.9995 was found for the linear calibration curve (n=2) of thalidomide with limits of quantitation of 0.222–21 mg/l. The method appeared to be feasible for pharmacokinetic studies with thalidomide.