Improving the binding capacity of Ni2+ decorated porous magnetic silica spheres for histidine-rich protein separation.

Biomagnetic immobilization of histidine-rich proteins based on the single-step affinity adsorption of transition metal ions continues to be a suitable practice as a cost effective and a up scaled alternative to the to multiple-step chromatographic separations. In our previous work, we synthesised Porous Magnetic silica (PMS) spheres by one-step hydrothermal-assisted modified-stöber method. The obtained spheres were decorated with Ni(2+) and Co(2+), and evaluated for the capture of a H6-Tagged green fluorescence protein (GFP-H6) protein. The binding capacity of the obtained spheres was found to be slightly higher in the case Ni(2+) decorated PMS spheres (PMSNi). However, comparing with commercial products, the binding capacity was found to be lower than the expected. In this way, the present work is an attempt to improve the binding capacity of PMSNi to histidine-rich proteins. We find that increasing the amount of Ni(2+) onto the surface of the PMS spheres leads to an increment of the binding capacity to GFP-H6 by a factor of two. On the other hand, we explore how the size of histidine-rich protein can affect the binding capacity comparing the results of the GFP-6H to those of the His-tagged α-galactosidase (α-GLA). Finally, we demonstrate that the optimization of the magnetophoresis parameters during washing and eluting steps can lead to an additional improvement of the binding capacity.

Design and characterization of Ni2+ and Co2+ decorated Porous Magnetic Silica spheres synthesized by hydrothermal-assisted modified-Stöber method for His-tagged proteins separation.

The complete elimination of enzymes from the reaction mixture and the possibility of its recycling for several rounds result in great benefits, allowing the reduction of the enzyme consumption and their usability in continuous processes. In this work, it is evaluated the capture of a H6-tagged green fluorescence protein (GFP-H6) on porous magnetic spheres using the Co(2+) and Ni(2+) affinity adsorption as a possible cost-effective and up-scaled alternative way for the immobilization of His-tagged proteins. For this purpose, Porous Magnetic Silica (PMS) spheres were synthesized by one-step hydrothermal-assisted modified-Stöber method. The obtained spheres have a homogenous size distribution of 400 nm diameter. The γ-Fe(2)O(3) nanoparticles are homogenously distributed in the silica matrix. The obtained PMS spheres have a saturation magnetization of about 10 emu/g. Magnetophoresis measurements show a total separation time of 16 min at 60 T/m. The obtained PMS spheres were successfully and homogenously decorated with Co(2+) and Ni(2+) and then evaluated for the capture of a GFP-H6 protein. The results were compared with the performance of the commercial beads Dynabeads® His-Tag Isolation & Pulldown.

Comparison of serologic assays for detection of antibodies against human herpesvirus 8.

Improvement of serologic assays for detection of antibodies against human herpesvirus 8 (HHV-8) is critical to better understand its epidemiology and biology. We produced the HHV-8 latent (ORF73) and lytic (ORF65, K8.1, and glycoprotein B) antigens in the Semliki Forest virus system and evaluated their performance in immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). These assays were compared with other latent antigen-based assays, including an IFA based on primary effusion lymphoma (PEL) cells and an ELISA based on bacterially expressed ORF73 antigen, as well as with other lytic antigen-based assays, including an IFA based on induced PEL cells, a commercial ELISA based on purified virions, and ELISAs based on K8.1- and ORF65-derived oligopeptides. We used a panel of 180 serum specimens obtained from three groups expected to have high, intermediate, and low HHV-8 prevalences. Using three different evaluation methods, we found that (i) the performances of the lytic antigen-based ELISAs were almost equivalent, (ii) the lytic antigen-based assays were more sensitive than the latent antigen-based assays, and (iii) in general, IFAs were more sensitive than ELISAs based on the same open reading frame. We also found that serum specimens from healthy individuals contained antibodies cross-reactive with HHV-8 glycoprotein B that can potentially cause false-positive reactions in lytic PEL-based IFAs. Although this is not a substantial problem in most epidemiologic studies, it may confound the interpretation of data in studies that require high assay specificity. Because the K8.1-based IFA provides sensitivity similar to that of lytic PEL-based IFAs and improved specificity, it can be a useful alternative to the PEL-based IFAs.

Cell lysis in Escherichia coli cultures stimulates growth and biosynthesis of recombinant proteins in surviving cells.

Cell growth and production of recombinant proteins in stationary phase cultures of Escherichia coli recover concomitantly with spontaneous lysis of a fraction of the ageing cell population. Further exploration of this event has indicated that sonic cell disruption stimulates both cell growth and synthesis of plasmid-encoded recombinant proteins, even in exponentially growing cultures. These observations indicate an efficient cell utilisation of released intracellular material and also that this capability is not restricted to extreme nutrient-starving conditions. In addition, the efficient re-conversion of waste cell material can be viewed as a potential strategy for an extreme exploitation of carbon sources and cell metabolites in production processes of both recombinant and non-recombinant microbial products.

Proteolytic digestion of bacterial inclusion body proteins during dynamic transition between soluble and insoluble forms.

Inclusion bodies formed by two closely related hybrid proteins, namely VP1LAC and LACVP1, have been compared during their building in Escherichia coli. Features of these proteins are determinant of aggregation rates and protein composition of the bodies, generating insoluble particles with distinguishable volume evolution. Interestingly, in LACVP1 and less perceptibly in VP1LAC bodies, an important fraction of the aggregated polypeptide is lost at a given stage of body construction. Stable degradation intermediates of the more fragile LACVP1 are concomitantly found embedded in the bodies. When recombinant protein synthesis is arrested in growing cells, the amount of aggregated protein drops while the amount of soluble protein undergoes a sudden rise before proteolysis. This indicates an architectural plasticity during the in vivo building of the studied inclusion bodies by a dynamic transition between soluble and insoluble forms of the recombinant proteins involved. During this transition, protease-sensitive polypeptides can suffer an efficient proteolytic attack and the resulting fragments further aggregate as inclusion body components.

Heat-inactivation of plasmid-encoded CI857 repressor induces gene expression from Ind- lambda prophage in recombinant Escherichia coli.

We have observed significant cell lysis upon temperature up-shift of recombinant Escherichia coli cultures harboring CI857-repressed lambda-based expression vectors. This event, that becomes evident about 30-40 min after the heat shock, takes place when using the lambda promoter system in Ind- lysogenic strains, but not in others commonly employed for recombinant gene expression. These results strongly suggest that the thermosensitive CI857 repressor, encoded by the expression vector, competes with CI Ind- molecules for binding to the prophage operator region, allowing for expression of lytic genes from the integrated Ind- viral genome upon temperature up-shift. Transcription of viral lytic genes does not include unspecific expression of a reporter sulA::lacZ gene fusion carried in the prophage genome. These results prompt, however, to carefully evaluate the limitations of expression systems based on pL/pR-CI857 in bacterial strains modified through lambda Ind- gene transfer vehicles.

Tolerance of Escherichia coli beta-galactosidase C-terminus to different-sized fusions.

The tolerance of the beta-galactosidase C-terminus to foreign protein fusions has been explored by using different-sized derivatives of the chimeric protein LACVP1. While the molecular mass of the partner domain shows a minor influence on protein toxicity for the producing E. coli cells, it dramatically affects the proteolytic susceptibility of the whole fusion. Surprisingly, the observed structural modulation of proteolysis is not an all-or-nothing process, but it exhibits a continuous effect concomitantly with the length of the fusion. The conformational effects caused by increasingly sized partners seem to progressively expose cryptic protease target sites, initiating a proteolytic cascade that dramatically reduces the yield of the recombinant protein.

Dynamics of in vivo protein aggregation: building inclusion bodies in recombinant bacteria.

Time-dependent aggregation of a plasmid-encoded beta-galactosidase fusion protein, VP1LAC, has been carefully monitored during its high-rate synthesis in Escherichia coli. Immediately after recombinant gene induction, the full-length form of the protein steadily accumulates into rapidly growing cytoplasmic inclusion bodies. Their volume increases during at least 5 h at a rate of 0.4 micron3 h-1, while the average density remains constant. Protein VP1LAC accounts for about 90% of the aggregated protein throughout the building process. Minor components, such as DnaK and GroEL chaperones, have been identified in variable, but low concentrations. The homogeneous distribution of inclusion bodies among the cell population and the coexistence of large, still growing bodies with newly appearing aggregates indicate that the aggregation cores are mutually exclusive, this fact being a main determinant of the in vivo dynamics of protein aggregation.

Numerical techniques and mathematical modelling for CI857-controlled gene expression and cell growth in recombinant E. coli.

Recombinant gene expression, monitored by beta-galactosidase activity, is studied in a pL, pR-CI857 plasmid expression system in temperature-induced E. coli batch cultures. The experimental procedure has been mathematically modelled, and the corresponding parameters are estimated from specific statistical and numerical methods, basically by using a global least-squares procedure under some constraints induced by the model. The numerical techniques proposed in this work act by accumulation of data coming from several runs of the modelled experiment, so that more accuracy is obtained in the parameter estimation. In particular, for the production process, an extra-model parameter depending on an indicator vector is introduced for each run of the experiment in order to globalize the data. The analysis of the data obtained leads to an integrated model for both cell growth and gene expression, which describes an asymmetric dynamics between culture growth and recombinant protein yield, and can serve to predict the maximal value of accumulated gene expression and the time required for it to be achieved at any age of the preinducing cell growth.

Plasmid maintenance in Escherichia coli recombinant cultures is dramatically, steadily, and specifically influenced by features of the encoded proteins.

A set of eight closely related plasmid constructs carrying CI857-controlled recombinant genes has been used as a model to study plasmid stability in Escherichia coli, in the absence of antibiotic selection. Plasmid loss rates and relative interdivision times of plasmid-bearing cells and plasmid-free cells have been analyzed throughout prolonged cultures. Whereas the calculated plasmid loss rates are not consistent for a given plasmid and set of conditions, the relative growth fitness of plasmid-bearing cells is highly reproducible. In the absence of gene expression, plasmid maintenance is influenced by the length of the cloned segment, the growth temperature, and the plasmid copy number, but not by the plasmid size. At high, inducing temperatures, the effects of the metabolic burden are eclipsed by the toxicity exhibited by the different proteins produced, which is determined by structural features. Despite the multifactorial nature of the negative pressures acting independently on plasmid-bearing cells, the relative cell fitness in a mixed cell population is very reproducible for a given vector, resulting in a monotonous spread of the plasmid-free cells in recombinant cultures.

The expression of recombinant genes from bacteriophage lambda strong promoters triggers the SOS response in Escherichia coli.

The production of several non-related heterologous proteins in recombinant Escherichia coli cells promotes a significant transcription of recA and sfiA SOS DNA repair genes. The activation of the SOS system occurs when the expression of plasmid-encoded genes is directed by the strong lambda lytic promoters, but not by IPTG-controlled promoters either at 37 or at 42 degrees C, and it is linked to an extensive degradation of the proteins after their synthesis. The triggering signal for the SOS response could be an important arrest of cell DNA replication observed within the first hour after the induction of recombinant gene expression. The stimulation of this DNA repair system can partially account for the toxicity exhibited by recombinant proteins on actively producing E. coli cells.

Limited in vivo proteolysis of aggregated proteins.

Degradation pathways of insoluble proteins have been analyzed in Escherichia coli by using a N-terminal beta-galactosidase fusion protein (VP1LAC) that aggregates immediately after its synthesis. In recombinant E. coli cells, lower molecular mass products, antigenically related to the entire fusion, accumulate together with the entire fusion. In absence of protein synthesis, the insoluble intact protein declines, suggesting that degradation of the recombinant protein also affects aggregated protein. Time course analysis of both soluble and insoluble cell fractions has revealed a limited proteolysis of the insoluble protein that removes the heterologous domain and permits the resulting beta-galactosidase fragments to refold and solubilize. Further extensive degradation occurs exclusively on soluble protein. The restricted proteolysis of misfolded, insoluble protein is the initiating event of a subsequent degradative pathway in which rate-limiting steps permit the accumulation of stable degradative intermediates.

Reversible activation of a cryptic cleavage site within E. coli beta-galactosidase in beta-galactosidase fusion proteins.

The VP60 capsid protein of rabbit haemorrhagic disease virus (60 kDa) has been fused to the C-terminus of beta-galactosidase and produced in E. coli from two related expression vectors. One of these vectors, carries a 429 bp DNA segment encoding the N-terminus peptide of VP60, and directs the synthesis of a larger fusion that contains the entire viral protein. Both fusion proteins are efficiently cleaved at a presumed trypsin-like target site within the carboxy moiety of beta-galactosidase (Arg 611-Thr 612), which is activated by the presence of the viral partner. In the larger fusion, VP60 is released by a cleavage within the linker region that affects about 10% of the chimeric proteins. In this situation, the resulting beta-galactosidase-like fragment recovers its natural proteolytic stability. These results prove that cryptic cleavage sites in beta-galactosidase can be efficiently activated in a fusion protein and suggest that this activation is based on reversible steric constraints generated by the fusion partner.

Antigenicity of a viral peptide displayed on beta-galactosidase fusion proteins is influenced by the presence of the homologous partner protein.

Several beta-galactosidase fusion proteins have been constructed containing the entire VP1 protein from foot-and-mouth disease virus (FMDV) [Corchero et al. (1996) J. Biotechnol. in press]. The antigenicity of the major immunodominant site A (13 amino acids in length) within the VP1 protein has been studied in competitive ELISA using a panel of seven monoclonal antibodies elicited against the whole virus and recognizing B-cell epitopes within this site. None of the fusion proteins is able to reproduce the antigenic profile of FMDV, all of them being less immunoreactive than the virus particles. On the other hand, significant differences in the reactivity of site A are displayed on the different fusion proteins, being for some antibodies about 10-fold. This indicates that the reactivity of a small peptide included in its natural place inside the heterologous domain can be significantly influenced by the position of the homologous partner in the fusion protein.

The position of the heterologous domain can influence the solubility and proteolysis of beta-galactosidase fusion proteins in E. coli.

The VP1 protein (23 kDa) of the foot-and-mouth disease virus has been produced in MC1061 and BL21 E. coli strains as beta-galactosidase fusion proteins, joined to either the amino and/or the carboxy termini of the bacterial enzyme. In BL21, devoid of La protease, all the recombinant fusion proteins are produced at higher yields than in MC1061, and occur mainly as inclusion bodies. The fusion of VP1 at the carboxy terminus yields a protease-sensitive protein whose degradation releases a stable, enzymatically active polypeptide indistinguishable from the native beta-galactosidase. On the contrary, when the same viral domain is fused to the amino terminus, the resulting chimeric protein is resistant to proteolysis even in the soluble form. These data demonstrate that the position of the heterologous domain in beta-galactosidase fusion proteins would not be irrelevant since it can dramatically influence properties of biotechnological interest such as solubility and proteolytic resistance.

A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli.

The 3D gene of foot-and-mouth disease virus encodes the viral RNA dependent RNA polymerase, also called virus infection associated (VIA) antigen, which is the most important serological marker of virus infection. This 3D gene from a serotype C1 virus has been cloned and overexpressed in Escherichia coli under the control of the strong lambda lytic promoters. The resulting 51 kDa recombinant protein has been shown to be immunoreactive with sera from infected animals. After induction of gene expression, an immediate and dramatic arrest of cell DNA synthesis occurs, similar to that produced by genotoxic doses of the drug mitomycin C. This effect does not occur during the production of either a truncated VIA antigen or other related and non-related viral proteins. The inhibition of DNA replication results in a subsequent induction of the host SOS DNA-repair response and in an increase of the mutation frequency in the surviving cells.

Mitomycin C stimulates thermally induced recombinant gene expression in Escherichia coli MC strains.

The effects of mitomycin C on C1857-controlled recombinant gene expression have been explored in E. coli cultures when the drug was added simultaneously to the thermal induction. A significantly improved yield of homologous, heterologous and chimeric fusion proteins was observed in E. coli MC1061 and GE864 (a MC4100 derivative) thermoinduced cells. This feature was not detected in other E. coli strains and does not involve a gene dosage mechanism but a strain-dependent stimulation of gene expression unrelated to the RecA protease activity.

Ammonium-mediated reduction of plasmid copy number and recombinant gene expression in Escherichia coli.

The effect of ammonium as a medium supplement on plasmid-encoded recombinant beta-galactosidase synthesis was explored in Escherichia coli cells during aerobic growth in complex medium. After induction, only doses of ammonium chloride below 1 g/L are able to transiently enhance the yield. However, the presence of nontoxic ammonium chloride concentrations of up to 10 g/L results in lower values of beta-galactosidase in a concentration-dependent fashion. A significant reduction in plasmid DNA content explains the decrease in the yield by a gene-dosage-involving mechanism.