Purification of recombinant rotavirus VP7 glycoprotein for the study of in vitro rotavirus-like particles assembly

Rotavirus VP7 is a glycoprotein that forms the viral capsid outerlayer and is essential to the correct assembly of triple-layered rotavirus-like particles (RLPs). In this work, a novel purification strategy was designed to allow obtaining highly pure monomeric VP7 required for the RLPs in vitro assembly. VP7 production kinetics in baculovirus-insect cells at cell concentration at infection (CCI) of 1x10(6)cellsmL(-1) was compared in terms of VP7/glycoprotein 64 (gp64) ratio at different multiplicity of infection (MOI). The best productivity was achieved at MOI of 0.1plaque forming unit (pfu)cell(-1) and time of harvest of 80h post-infection. After preliminary clarification steps, the proteins eluted from Concanavalin A were concentrated and loaded onto size exclusion chromatography. The polishing step was anion exchange chromatography with Mono Q. The high resolution of this column resulted in separation of monomers from dimers of VP7. Overall, the purification protocol yielded high level of purity (>90%). Purified VP7 was characterized by MALDI-TOF mass spectrometry and SDS-capillary gel electrophoresis. The MW and apparent MW were determined as 31.6 and 39kDa, respectively, confirming the efficacy of the proposed purification strategy that now enables RLPs assembly studies.     

Use of recombinant rotavirus VP6 nanotubes as a multifunctional template for the synthesis of nanobiomaterials functionalized with metals

The structural characteristics and predefined constant size and shape of viral assemblies make them useful tools for nanobiotechnology, in particular as scaffolds for constructing highly organized novel nanomaterials. In this work it is shown for the first time that nanotubes formed by recombinant rotavirus VP6 protein can be used as scaffolds for the synthesis of hybrid nanocomposites. Rotavirus VP6 was produced by the insect cell‐baculovirus expression vector system. Nanotubes of several micrometers in length and various diameters in the nanometer range were functionalized with Ag, Au, Pt, and Pd through strong (sodium borohydride) or mild (sodium citrate) chemical reduction. The nanocomposites obtained were characterized by transmission electron microscopy (TEM), high‐resolution TEM (HRTEM) with energy dispersive spectroscopy (EDS), dynamic light scattering, and their characteristic plasmon resonance. The outer surface of VP6 nanotubes had intrinsic affinity to metal deposition that allowed in situ synthesis of nanoparticles. Furthermore, the use of preassembled recombinant protein structures resulted in highly ordered integrated materials. It was possible to obtain different extents and characteristics of the metal coverage by manipulating the reaction conditions. TEM revealed either a continuous coverage with an electrodense thin film when using sodium citrate as reductant or a discrete coverage with well‐dispersed metal nanoparticles of diameters between 2 and 9 nm when using sodium borohydride and short reaction times. At long reaction times and using sodium borohydride, the metal nanoparticles coalesced and resulted in a thick metal layer. HRTEM‐EDS confirmed the identity of the metal nanoparticles. Compared to other non‐recombinant viral scaffolds used until now, the recombinant VP6 nanotubes employed here have important advantages, including a longer axial dimension, a dynamic multifunctional hollow structure, and the possibility of producing them massively by a safe and efficient bioprocess. Such characteristics confer important potential applications in nanotechnology to the novel nanobiomaterials produced here. Biotechnol. Bioeng. 2009; 104: 871–881. © 2009 Wiley Periodicals, Inc.     

Plasmonic Circular Dichroism Study of DNA–Gold Nanoparticles Bioconjugates

Plasmonic circular dichroism (CD) responses of hybrid nanostructures containing noble metal nanoparticles and chiral molecules have received increasing interest with various applications in nanophotonics. Chiral biomolecules show strong CD signals typically found in the ultraviolet region, whereas, in the visible range, they produce a weak signal. Strengthening the CD signal in the visible region is of high importance, which could be achieved through fabrication of novel hybrid nanostructures. Herein, gold nanoparticles (GNPs) have been assembled via DNA linker to investigate the possibility of enhancing plasmonic CD signal in the visible range. DNA-linked assemblies with pre- and postannealed conditions were characterized by ultraviolet–visible spectroscopy, dynamic light scattering (DLS), and CD spectropolarimetry. In the presence of DNA linker with sticky ends, the aggregation phenomenon was traced by red shifts of surface plasmon resonance of nanoparticles. Time-dependent hybridization of single-stranded “sticky ends” with DNA-conjugated GNPs and increased probability of hydrogen bond formation lead to enhancement of CD signals in the ultraviolet region. Complexation of biomolecule and nanoparticle assemblies induced enhanced CD signals in the visible range, which was noticed both before and after purification. DLS characterization of the assemblies also confirmed the difference in the size of aggregates, which could be controlled by the linker molecules. This investigation encourages possibility of utilizing plasmonic CD technique as a tool for tracing fabricated nanostructure assemblies with enhanced characterization possibility.     

Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellular heptameric fusion protein

An integrated bioprocess has been developed suitable for production of recombinant peptides using a gene multimerization strategy and site-specific cleavage of the resulting gene product. The process has been used for production in E. coli of the human proinsulin C-peptide via a fusion protein BB-C7 containing seven copies of the 31-residues C-peptide monomer. The fusion protein BB-C7 was expressed at high level, 1.8 g l(-1), as a soluble gene product in the cytoplasm. A heat treatment procedure efficiently released the BB-C7 fusion protein into the culture medium. This step also served as an initial purification step by precipitating the majority of the host cell proteins, resulting in a 70% purity of the BB-C7 fusion protein. Following cationic polyelectrolyte precipitation of the nucleic acids and anion exchange chromatography, native C-peptide monomers were obtained by enzymatic cleavage at flanking arginine residues. The released C-peptide material was further purified by reversed-phase chromatography and size exclusion chromatography. The overall yield of native C-peptide at a purity exceeding 99% was 400 mg l(-1) culture, corresponding to an overall recovery of 56%. The suitability of this process also for the production of other recombinant proteins is discussed.     

Downstream processing of triple layered rotavirus like particles

Rotavirus like particles (RLPs) constitute a potential vaccine for the prevention of rotavirus disease, responsible for the death of more than half a million children each year. Increasing demands for pre-clinical trials material require the development of reproducible, scaleable and cost-effective purification strategies as alternatives to the traditional laboratory scale CsCl density gradient ultracentrifugation methods commonly used for the purification of these complex particles. Self-assembled virus like particles (VLPs) composed by VP2, VP6 and VP7 rotavirus proteins (VLPs 2/6/7) were produced in 5l scale using the insect cells/baculovirus expression system. A purification process using depth filtration, ultrafiltration and size exclusion chromatography as stepwise unit operations was developed. Removal of non-assembled rotavirus proteins, concurrently formed particles (RLP 2/6), particle aggregates and products of particle degradation due to shear was achieved. Particle stability during storage was studied and assessed using size exclusion chromatography as an analytical tool. Formulations containing either glycerol (10% v/v) or trehalose (0.5 M) were able to maintain 75% of intact triple layered VLPs, at 4 degrees C, up to 4 months. The overall recovery yield was 37% with removal of 95% of host cell proteins and 99% of the host cell DNA, constituting a promising strategy for the downstream processing of other VLPs.     

Isolation and characterization of a 1,4-beta-D-glucan glucohydrolase from the yeast, Torulopsis wickerhamii

1,4-beta-D-Glucan glucohydrolase (exo-1,4-beta-D-glucosidase) (EC 3.2.1.74) was isolated from growth supernatants of Torulopsis wickerhamii and was subjected to hydrodynamic, optical (CD), and kinetic analysis after purification to homogeneity by ammonium sulfate precipitation, size exclusion chromatography, ion exchange chromatography, and isopycnic banding centrifugation in cesium chloride. The last step was found to separate the enzyme from strongly associating, high molecular weight polysaccharide. Enzyme homogeneity was established by isoelectric focusing, sodium dodecyl sulfate-gel electrophoresis, and analytical high performance size exclusion chromatography using dual detection. The native exo-1,4-beta-D-glucosidase was found to be a dimer of 151,000 +/- 21,100 daltons by high performance size exclusion chromatography and 143,600 +/- 1,800 daltons by sedimentation equilibrium. The enzyme has a 12% linked carbohydrate content (mostly mannose) and no essential metal ions. Hydrolysis of p-nitrophenyl-beta-D-glucopyranoside was found to be optimal at pH 4.25 and 50 degrees C. The enzyme was found to produce beta-D-glucose from cellodextrins (indicating retention of anomeric configuration during hydrolysis) and demonstrated depolymerization from the non-reducing polymer terminus. The enzyme followed competitive type inhibition with p-nitrophenyl-beta-D-glucopyranoside as substrate and demonstrated high values of Ki for D-glucose and D-cellobiose inhibition (190 and 230 mM, respectively). The exo-1,4-beta-D-glucosidase was found to hydrolyze cellotetraose more rapidly than D-cellobiose and aryl-beta-D-glycosides more rapidly than all other substrates. Low levels of activity were found for the polymeric substrates beta-glucan (yeast cell walls), Avicel, and Walseth cellulose. Although this enzyme demonstrates broad disaccharide substrate specificity, a characteristic common to beta-D-glucosidases from many sources, the ability to hydrolyze higher cellodextrins more rapidly than cellobiose renders this enzyme the first exo-1,4-beta-D-glucosidase purified from yeast.     

Characterization of VP22 in herpes simplex virus-infected cells

We examine biochemical characteristics of the herpes simplex virus (HSV) tegument protein VP22 by gel filtration, glycerol sedimentation, and chemical cross-linking experiments and use time course radiolabeling and immunoprecipitation assays to analyze its synthesis and interaction with other infected-cell proteins. VP22 was expressed as a delayed early protein with optimal synthesis requiring DNA replication. In immunoprecipitation assays, VP22 was found in association with several additional proteins including VP16 and a kinase activity likely to be that of UL13. Furthermore, in sizing chromatography experiments, VP22 was present in several higher-order complexes in infected cells. From gel filtration analysis the major form of VP22 migrated with a molecular mass of approximately 160 kDa, consistent with its presence as a tetramer, or a dimer complexed with other proteins, with a fraction of the protein migrating at larger molecular mass. In vitro-synthesized VP22 sedimented in a size range consistent with a mixture of tetramers and dimers. Short N- or C-terminal deletions resulted in migration almost exclusively as dimers, indicating that VP22, in the absence of additional virus-encoded proteins, could form higher-order assemblies, most likely tetramers, but that both N-and C-terminal determinants were required for stabilizing such assemblies. Consistent with this we found that isolated proteins encompassing either the N-terminal or C-terminal region of VP22 sedimented as dimers, and that the purified C-terminal domain could be cross-linked into dimeric structures. These results are discussed with regard to possible virus and host interactions involved in VP22 recruitment into virus particles.     

Over-expression, purification and characterization of the oligomerization dynamics of an invertebrate mitochondrial creatine kinase

Mitochondrial creatine kinase (MtCK) plays a central role in energy homeostasis within cells that display high and variable rates of ATP turnover. Vertebrate MtCKs exist primarily as octamers but readily dissociate into constituent dimers under a variety of circumstances. MtCK is an ancient protein that is also found in invertebrates including sponges, the most primitive of all multi-cellular animals. We have cloned, expressed, and purified one of these invertebrate MtCKs from a marine polychaete worm, Chaetopterus variopedatus (CVMtCK). Size exclusion chromatography and dynamic light scattering (DLS) were used to characterize oligomeric state in comparison with that of octameric chicken sarcomeric isoform (SarMtCK). At protein concentrations >1 mg/ml, CVMtCK was predominantly octameric (>90%). When diluted to 0.1 mg/ml, CVMtCK dissociated into dimers much more rapidly than SarMtCK when observed under identical conditions. The rate of dissociation for both MtCKs increased as temperature rose from 2 to 28 degrees C, and in CVMtCK, fell at higher incubation temperatures. The fraction of octameric CVMtCK at equilibrium increased with temperature and then fell. Temperature transition studies showed that octamers and dimers were rapidly interconvertible on a similar time scale. Importantly, when CVMtCK was converted to the transition state analog complex (TSAC), both size exclusion chromatography and DLS showed that there was minimal dissociation of octamers into dimers while SarMtCK octamers were highly unstable as the TSAC. These results clearly show distinct differences in octamer stability between CVMtCK and SarMtCK, which could impact function under physiological circumstances. Furthermore, the large yield of recombinant protein and high stability of CVMtCK in the TSAC suggest that this protein might be a good target for crystallization efforts.     

Structural polymorphism of the major capsid protein of rotavirus

Rotaviruses are important human pathogens with a triple-layered icosahedral capsid. The major capsid protein VP6 is shown here to self-assemble into spherical or helical particles mainly depending upon pH. Assembly is inhibited either by low pH (<3.0) or by a high concentration (>100 mM) of divalent cations (Ca(2+) and Zn(2+)). The structures of two types of helical tubes were determined by electron cryomicroscopy and image analysis to a resolution of 2.0 and 2.5 nm. In both reconstructions, the molecular envelope of VP6 fits the atomic model determined by X-ray crystallography remarkably well. The 3-fold symmetry of the VP6 trimer, being incompatible with the helical symmetry, is broken at the level of the trimer contacts. One type of contact is maintained within all VP6 particles (tubes and virus), strongly suggesting that VP6 assemblies arise from different packings of a unique dimer of trimers. Our data show that the protonation state and thus the charge distribution are important switches governing the assembly of macromolecular assemblies.     

Purification of recombinant HIV-1 protease.

A method is described to purify recombinant HIV-1 protease from soluble extracts of Escherichia coli. The isolation involves QAE-Sepharose anion exchange chromatography, hexyl agarose hydrophobic interaction chromatography, MonoS cation exchange chromatography, and Superose 6 size exclusion chromatography. Approximately 100 micrograms of protease was obtained from 18 g E. coli paste. The protein was judged to be homogeneous due to the presence of a single band on a silver-stained SDS polyacrylamide gel.     

Using precipitation by polyamines as an alternative to chromatographic separation in antibody purification processes

Polyamine precipitation conditions for removing host cell protein impurities from the cell culture fluid containing monoclonal antibody were studied. We examined the impact of polyamine concentration, size, structure, cell culture fluid pH and ionic strength. A 96-well microtiter plate based high throughput screening method was developed and used for evaluating different polyamines. Polyallylamine, polyvinylamine, branched polyethyleneimine and poly(dimethylamine-co-epichlorohydrin-ethylenediamine) were identified as efficient precipitants in removing host cell protein impurities. Leveraging from the screening results, we incorporated a polyamine precipitation step into a monoclonal antibody purification process to replace the Protein A chromatography step. The optimization of the overall purification process was performed by taking the mechanisms of both precipitation and chromatographic separation into account. The precipitation-containing process removed a similar amount of process-related impurities, including host cell proteins, DNA, insulin and gentamicin and maintained similar product quality in respect of size and charge variants to chromatography based purification. Overall recovery yield was comparable to the typical Protein A affinity chromatography based antibody purification process.     

pcD-awte候选疟疾DNA疫苗制备及其质量相关性分析

本实验室构建的疟疾DNA疫苗经动物试验表明具有很好的免疫原性,为申请临床试验,进行了制备工艺的研究。本研究将含pcD-awte质粒的大肠杆菌DH5α在发酵罐中发酵培养,碱裂解法粗提质粒,再依次通过Sepharose 6FF分子筛层析、Plasmidselect 亲硫吸附层析和Source 30Q离子交换层析精制获得质粒纯品,并对纯品进行质量分析。结果每升培养液可获得质粒纯品43.9mg,质量符合Ferreira等推荐的药用标准。     

Characterization of the major egg glycolipoproteins from the perivitellin fluid of the apple snail Pomacea canaliculata

Ovorubin and PV2 are the major lipoglycocarotenoproteins present in the perivitellus of the freshwater snail eggs of Pomacea canaliculata, a rapidly expanding rice field pest. We have previously characterized these two particles regarding their lipid and protein compositions, their synthesis and tissular distribution, and their contributions of energy and structural precursors for the developing embryo. In the present study, we have characterized the glycosidic moieties associated to these perivitellines. Both proteins were isolated from egg homogenates by ultracentrifugation, and high performance liquid chromatography (HPLC) using anionic exchange and size exclusion columns. Total carbohydrates accounted for 17.8% and 2.5% (w/w) of the apparent molecular mass of ovorubin and PV2, respectively. Analysis by size exclusion chromatography showed that the amount of O-linked oligosaccharides is higher than that of the N-linked species (59% and 67% w/w of total carbohydrates of ovorubin and PV2, respectively). Glycosylation patterns were determined by a set of biotinilated lectins onto blotted purified proteins. Lectin affinities confirmed the presence of aspargine-linked carbohydrates, probably of hybrid and high mannose types. Jacaline affinity suggested the presence of O-linked residues derived from the T-antigen. Total carbohydrate composition determined by gas liquid chromatography (GLC) showed that mannose was the major monosaccharide in both perivitellins followed by GlcNAc and Gal in ovorubin, and Gal and GlcNAc in PV2. Only one fatty acid (22:1 n-9) accounted for 46% and 56% of the fatty acids present in ovorubin and PV2, respectively. Carbohydrate role on these reserve proteins during embryogenesis of the apple snail is discussed.     

Continuous removal of protein aggregates by annular chromatography

The removal of polymeric proteins from their monomers is a frequently encountered separation task, especially in the polishing step of therapeutic proteins. Continuous separation of protein polymers from monomers by annular chromatography using size exclusion chromatography has been studied regarding the resolution, recovery, fouling, and productivity and has been compared to conventional chromatography. An IgG preparation rich in aggregates was used as a model protein mixture. Under conditions that maximized the throughput, the polymers could be separated from the monomers, but baseline separation could not be achieved. Baseline separation was also not possible in batch mode using equivalent conditions, which was also confirmed by computer simulation. For separation of the aggregates from the product the entire available separation space (360 degrees ) was indispensable. Therefore only cyclic, discontinuous regeneration could be carried out. Loading was identified as a critical step, since the concentrated protein solution evaded into the headspace instead of migrating into the gel where viscous fingering often occurs in conventional chromatography. The productivity of annular chromatography was two times higher than that of the conventional batch chromatography, and the buffer consumption was reduced to half the conventional value. These two benefits are especially important for protein separation processes that suffer from low loadability, such as size exclusion chromatography. We have demonstrated that size exclusion can be performed on an industrial scale when it is run continuously with the aid of a pressurized annular chromatograph.     

Expression of Rotavirus Capsid Protein VP6 in Transgenic Potato and Its Oral Immunogenicity in Mice

Murine rotavirus gene six encoding the 41 kDa group specific capsid structural protein VP6 was stably inserted into the Solanum tuberosum genome by Agrobacterium tumefaciens mediated transformation. The molecular mass of plant synthesized VP6 capsid protein determined by immunoblot was similar to the size of both purified virus VP6 monomeric peptides and partially assembled virus-like particles. The amount of VP6 protein synthesized in transgenic potato leaf and tuber was determined by enzyme-linked immunosorbent assay to be approximately 0.01% of total soluble protein. Oral immunization of CD-1 mice with transformed potato tuber tissues containing VP6 capsid protein generated measurable titers of both anti-VP6 serum IgG and intestinal IgA antibodies. The presence of detectable humoral and intestinal antibody responses against the rotavirus capsid protein following mucosal immunization provides an optimistic basis for the development of edible plant vaccines against enteric viral pathogens.     

High-performance liquid chromatographic method for the determination of insulin synthesis in biological systems

This paper reports a two-step high-performance liquid chromatographic procedure which permits the study of the incorporation of [³H]leucine into insulin in biological systems. The first step of the procedure was size exclusion chromatography, performed on a GPC-100 column, which was eluted with 0.1 M KH₂PO₄—methanol (9:1, v/v). By this step the bulk of both protein and radioactivity was separated from tritiated insulin. The second step, which employs reversed-phase chromatography on an octadecylsilyl column, permits the separation of insulin from other contaminants by means of a linear gradient of acetonitrile. This simple and reproducible method was employed to test insulin synthesis in cultured human retinoblastoma Y79 cells.     

Preclinical manufacture of anti-HER2 liposome-inserting, scFv-PEG-lipid conjugate. 2. Conjugate micelle identity, purity, stability, and potency analysis

Analytical methods optimized for micellar F5cys-MP-PEG(2000)-DPSE protein-lipopolymer conjugate are presented. The apparent micelle molecular weight, determined by size exclusion chromatography, ranged from 330 to 960 kDa. The F5cys antibody and conjugate melting points, determined by differential scanning calorimetry, were near 82 degrees C. Traditional methods for characterizing monodisperse protein species were inapplicable to conjugate analysis. The isoelectric point of F5cys (9.2) and the conjugate (8.9) were determined by capillary isoelectric focusing (cIEF) after addition of the zwitterionic detergent CHAPS to the buffer. Conjugate incubation with phospholipase B selectively removed DSPE lipid groups and dispersed the conjugate prior to separation by chromatographic methods. Alternatively, adding 2-propanol (29.4 vol %) and n-butanol (4.5 vol %) to buffers for salt-gradient cation exchange chromatography provided gentler, nonenzymatic dispersion, resulting in well-resolved peaks. This method was used to assess stability, identify contaminants, establish lot-to-lot comparability, and determine the average chromatographic purity (93%) for conjugate lots, described previously. The F5cys amino acid content was confirmed after conjugation. The expected conjugate avidity for immobilized HER-2/neu was measured by bimolecular interaction analysis (BIAcore). Mock therapeutic assemblies were made by conjugate insertion into preformed doxorubicin-encapsulating liposomes for antibody-directed uptake of doxorubicin by HER2-overexpressing cancer cells in vitro. Together these developed assays established that the manufacturing method as described in the first part of this study consistently produced F5cys-MP-PEG(2000)-DSPE having sufficient purity, stability, and functionality for use in preclinical toxicology investigations.