Biomedical subjects

Ario de Marco

Publications and source records attributed to Ario de Marco.

14 recordsLinked to original sources

PubMed2006

The evaluation of the factors that cause aggregation during recombinant expression in E. coli is simplified by the employment of an aggregation-sensitive reporter.

BACKGROUND: The yields of soluble recombinant proteins expressed in bacteria are often low due to the tendency of the heterologous proteins to form aggregates. Therefore, aggregation reporters have been envisaged to simplify the comparison among different expression conditions and to speed up the identification of suitable protocols that improve the solubility. The probe we used is composed by an IbpAB promoter specifically activated by protein aggregates fused to a sequence coding the beta-galactosidase, the activity of which becomes, therefore, indicative of the aggregation degree. RESULTS: The collected data show that the probe is reliable in terms of reproducibility inside a range of experimental conditions and faster and more sensitive than the analysis methods based on SDS-PAGE and successive western blot. The beta-galactosidase probe was useful to identify which parameters could influence the aggregation of the model proteins and to set up an optimized expression protocol. The effect of growth temperature, induction modality, co-expression with molecular chaperones and addition of osmolytes on the accumulation of aggregates were evaluated following the beta-galactosidase activity. Interestingly, a significant correlation was observed between estimated decreased aggregation and higher yields of soluble protein. We also compared a set of expression vectors with various regulative features and found that the single characteristics, like promoter, copy number or polymerase, were not relevant for controlling the recombinant protein aggregation whilst the crucial factor resulted being the total expression rate of the system. CONCLUSION: The aggregation reporter used in our experiments represents a useful tool to evaluate the different factors that can be modulated to optimize a recombinant expression protocol. Furthermore, the rapid estimation of the aggregation degree enables to discriminate this from other causes responsible for scarce recombinant yields.

Journal Article↗

PubMed2006

Induced fit of passenger proteins fused to Archaea maltose binding proteins.

Maltose binding proteins (MBPs) are used as carriers for improving the solubility of passenger proteins. Our results indicate that the higher solubility of the fusions correlates with their elevated heat stability. Fusions of the otherwise thermo-sensitive GFP with MBPs from Archaea, but not GST-GFP and Escherichia coli MBP-GFP, maintained their fluorescence and structure after 10min incubation at 100 degrees C and could be purified by heating the bacteria lysate, with yields even higher than those obtained using metal affinity chromatography. Furthermore, only correctly folded proteins could stand the heating treatment and, therefore, the heat-purification method can be used as a quality control step to select homogeneous monodispersed material whereas soluble aggregates are removed by precipitation.

Archaeal Proteins↗

PubMed2006

Automated protein analysis by online detection of laser-induced fluorescence in slab gels and 3-D geometry gels.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) still remains the most reliable and comprehensive analytical method for the evaluation of protein extracts. However, conventional SDS-PAGE is time-consuming and, thus, unpractical if several tens or hundreds of samples must be examined. We show that SDS-PAGE protein analysis can be automated using slab gel DNA sequencers and compare the instrument's performance with conventional SDS-PAGE in terms of resolution, sensitivity and sample capacity. Labeled protein bands are detected online by laser-induced fluorescence (LIF) and the acquired signals are electronically stored for further processing, avoiding gel staining and scanning. Appropriate software allows immediate display of recorded data and convenient evaluation. The method provides a higher sensitivity and dynamic range than conventional Coomassie-stained gels and the resolution of proteins with different masses is independent of the polyacrylamide concentration. Internal markers can also be used for direct quantification and assignment of the molecular masses. Additionally, we present a novel electrophoresis instrument for the simultaneous separation and online LIF detection of all samples of a microtiterplate in parallel lanes in a 3-D geometry gel cylinder. The specific gel thermostatting concept prevents irregular sample migration (smiling) and improves the reproducibility and comparability of individual separation patterns. In combination with the expected large capacity of 384 or 1,536 samples, this makes the instrument a valuable tool for high-throughput comparative screening applications.

Automation↗

PubMed2005

Simplified screening for the detection of soluble fusion constructs expressed in E. coli using a modular set of vectors.

BACKGROUND: The solubility of recombinant proteins expressed in bacteria is often disappointingly low. Several strategies have been developed to improve the yield and one of the most common strategies is the fusion of the target protein with a suitable partner. Despite several reports on the successful use of each of these carriers to increase the solubility of some recombinant proteins, none of them was always successful and a combinatorial approach seems more efficient to identify the optimal combination for a specific protein. Therefore, the efficiency of an expression system critically depends on the speed in the identification of the optimal combination for the suitable fusion candidate in a screening process. This paper describes a set of expression vectors (pETM) designed for rapid subcloning, expression and subsequent purification using immobilized metal affinity chromatography (IMAC). RESULTS: A single PCR product of two Yellow Fluorescent Proteins (EYFPs) was cloned into 18 vectors comprising identical restriction sites and varying fusion partners as well as differing protease recognition sites. After a small-scale expression, the yields of the different constructs were compared using a Coomassie stained SDS-polyacrylamide gel and the results of this preliminary screening were then confirmed by large-scale purification. The yields were calculated and the stability of the different constructs determined using three independent conditions. The results indicated a significant correlation between the length and composition of non-native amino acid tails and stability. Furthermore, the buffer specificity of TEV and 3C proteases was tested using fusion proteins differing only in their protease recognition sequence, and a His-GST-EYFP construct was employed to compare the efficiency of the two alternative affinity purification methods. CONCLUSION: The experiments showed that the set of pETM vectors could be used for the rapid production of a large array of different constructs with specific yield, stability, and cleavage features. Their comparison allowed the identification of the optimal constructs to use for the large-scale expression. We expect that the approach outlined in this paper, i.e. the possibility to obtain in parallel fusion products of the target protein with different partners for a preliminary evaluation, would be highly beneficial for all them who are interested in the rapid identification of the optimal conditions for protein expression.

Journal Article↗

PubMed2005

Nup155 regulates nuclear envelope and nuclear pore complex formation in nematodes and vertebrates.

Nuclear envelope (NE) formation during cell division in multicellular organisms is a central yet poorly understood biological process. We report that the conserved nucleoporin Nup155 has an essential function in NE formation in Caenorhabditis elegans embryos and in Xenopus laevis egg extracts. In vivo depletion of Nup155 led to failure of nuclear lamina formation and defects in chromosome segregation at anaphase. Nup155 depletion inhibited accumulation of nucleoporins at the nuclear periphery, including those recruited to chromatin early in NE formation. Electron microscopy analysis revealed that Nup155 is also required for the formation of a continuous nuclear membrane in vivo and in vitro. Time-course experiments indicated that Nup155 is recruited to chromatin at the time of NE sealing, suggesting that nuclear pore complex assembly has to progress to a relatively late stage before NE membrane assembly occurs.

Animals↗

PubMed2005

Fusion tags and chaperone co-expression modulate both the solubility and the inclusion body features of the recombinant CLIPB14 serine protease.

Chaperone co-expression and the fusion to different tags were used to modify the aggregation pattern of the putative serine protease CLIPB14 precipitated in Escherichia coli inclusion bodies. A set of common tags used in expression vectors has been selected, as well as two bacterial strains over-expressing the chaperones GroELS and ibpA/B, respectively. The presence of the fused tags resulted in an improved solubility of CLIPB14 but also in a higher presence of contaminants in the inclusion bodies, while chaperone co-expression promoted the binding of all the chaperone machinery involved into the disaggregation to the CLIPB14. Furthermore, each tag influenced in a specific manner the re-aggregation of the denatured CLIPB14 constructs during urea dilution and the preliminary trials indicated that the CLIPB14 fusions with higher homogeneity and lower re-aggregation rate were the optimal candidates for refolding assays. In conclusion, it is possible to tune the quality of the inclusion bodies by choosing the suitable combination of tag and chaperone co-expression that minimize the non-productive side reactions during refolding.

Animals↗

PubMed2005

Characterization of the aggregates formed during recombinant protein expression in bacteria.

BACKGROUND: The first aim of the work was to analyze in detail the complexity of the aggregates formed upon overexpression of recombinant proteins in E. coli. A sucrose step gradient succeeded in separating aggregate subclasses of a GFP-GST fusion protein with specific biochemical and biophysical features, providing a novel approach for studying recombinant protein aggregates. RESULTS: The total lysate separated into 4 different fractions whereas only the one with the lowest density was detected when the supernatant recovered after ultracentrifugation was loaded onto the sucrose gradient. The three further aggregate sub-classes were otherwise indistinctly precipitated in the pellet. The distribution of the recombinant protein among the four subclasses was strongly dependent on the DnaK availability, with larger aggregates formed in Dnak- mutants. The aggregation state of the GFP-GST recovered from each of the four fractions was further characterized by examining three independent biochemical parameters. All of them showed an increased complexity of the recombinant protein aggregates starting from the top of the sucrose gradient (lower mass aggregates) to the bottom (larger mass aggregates). These results were also confirmed by electron microscopy analysis of the macro-structure formed by the different aggregates. Large fibrils were rapidly assembled when the recombinant protein was incubated in the presence of cellular extracts, but the GFP-GST fusion purified soon after lysis failed to undergo amyloidation, indicating that other cell components probably participate in the active formation of large aggregates. Finally, we showed that aggregates of lower complexity are more efficiently disaggregated by a combination of molecular chaperones. CONCLUSION: An additional analytical tool is now available to investigate the aggregation process and separate subclasses by their mass. It was possible to demonstrate the complexity of the aggregation pattern of a recombinant protein expressed in bacteria and to characterize biochemically the different aggregate subclasses. Furthermore, we have obtained evidence that the cellular environment plays a role in the development of the aggregates and the problem of the artifact generation of aggregates has been discussed using in vitro models. Finally, the possibility of separating aggregate fractions with different complexities offers new options for biotechnological strategies aimed at improving the yield of folded and active recombinant proteins.

Bacterial Proteins↗

PubMed2005

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel.

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.

Bacterial Proteins↗

PubMed2005

Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones.

When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock-inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.

Animals↗

PubMed2004

A step ahead: combining protein purification and correct folding selection.

The success of recombinant protein expression seems unpredictable and even good yields of soluble proteins do not guarantee the correct folding. The search for soluble constructs can be performed by exploiting libraries and speeded up by automation, but these approaches are money and time consuming and the tags used for affinity purification can mask the real stability of the target proteins. The ideal purification protocol would include the structure quality control. A recent paper commented in this article describes a phage-display method to screen for antibodies that are able to re-fold after heat-denaturation and can be selectively affinity-purified only if monodispersed. It turned out that the proteins with high recovery performance after heat-shock were also suitable for efficient recombinant expression.

Editorial↗

PubMed2004

The solubility and stability of recombinant proteins are increased by their fusion to NusA.

The new bacterial vector pETM60 enables the expression of His-tagged recombinant proteins fused to the C-terminus of NusA through a TEV protease recognition sequence. Three sequences coding for two protein domains (Xklp3A and Tep3Ag) and one membrane-bound viral protein (E8R) could not be expressed in a soluble form in bacteria. Their GST-fusions were mostly soluble but quickly degraded during purification. The same sequences cloned in pETM60 were efficiently purified by metal affinity and recovered soluble after the removal of the fusion partner. The NusA-fused constructs enabled to yield 13-20mg of fusion protein per litre of culture and 2.5-5mg of pure protein per litre of culture. Structural analysis indicated that the purified proteins were monodispersed and correctly folded. NusA has been used to raise antibodies that have been successfully used for Western blot and immunoprecipitation of NusA fusion proteins.

Drug Stability↗

PubMed2004

Bacteria co-transformed with recombinant proteins and chaperones cloned in independent plasmids are suitable for expression tuning.

The efficient over-expression of several recombinant proteins in the same bacterial cell is usually prevented due to metabolic limitations. Nevertheless, the possibility to co-produce high amounts of the sub-units of a complex or to express a wide set of chaperones and foldases could be technologically very useful. We developed a system based on three vectors. Two are under IPTG regulation and enable the recombinant expression of six chaperones, the third one is arabinose-inducible and harbours the sequence for the target protein. In such a way the independent induction and the level of expression of both chaperones and target protein is possible. The data show that the expression leakage from pET vectors was prevented by the introduction of further plasmids in the cell and that the recombinant proteins compete for their expression. In fact, the high rate induction of one of them could switch off the accumulation of the other recombinant proteins. The first information was used to maximise the expression of toxic proteins while the cross-inhibition among recombinant proteins was exploited to modulate and optimise the target protein expression and to induce the chaperone-assisted in vivo re-folding of aggregated target protein.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2004

Recombinant proteins fused to thermostable partners can be purified by heat incubation.

We developed a protocol for the fast purification of small proteins and peptides using heat incubation as the first purification step. The proteins are expressed from a new bacterial expression vector (pETM-90) fused to the C-terminus of thermostable Ftr from Methanopyrus kandleri. The vector further contains a 6xHis-tag to allow immobilised metal ion affinity purification and a TEV protease cleavage site to enable the removal of the His-tag and fusion partner. Heat incubation induces the specific denaturation and precipitation of the Escherichia coli proteins but not of the thermostable fusion protein. Using the fusion construct and the heat incubation protocol a number of fusion proteins were purified to near homogeneity. The thermostability was ensured when Ftr had a molecular weight higher than twice the target protein. The obtained purification yields were similar and, in some cases, even higher than the ones obtained by affinity purification with the same Ftr-fusion proteins or the same target proteins fused to other often used partners such as NusA, GST, or DsbA. The protocol does not depend on a specific thermostable protein as was shown by the exchange of Ftr for M. kandleri Mtd. Purification by heat incubation is a fast and inexpensive alternative to chromatographic techniques, particularly suitable for the production of antigenic sequences for which the loss of native structure is not detrimental. We proved that it can be easily automated.

Amino Acid Sequence↗

PubMed2003

Correct identification of the chloroplastic protoporphyrinogen IX oxidase N-terminus places the biochemical data in frame.

Maize (Zea mays) protoporphyrinogen IX oxidase (PPO: EC 1.3.3.4) possesses a chloroplast transit peptide (CTP) that delivers the enzyme into the chloroplast. The cleavage site yielding the mature protein was predicted by using the ChloroP software and by comparing conserved regions of the available plant PPO sequences. In parallel, the processed NH(2)-terminus of native PPO was identified experimentally by microsequencing the immunoprecipitated plant PPO from maize etioplasts. The cleavage sites identified using the bioinformatic approaches did not match the experimental result. The three sequences have been cloned and expressed in bacteria and their kinetics were compared in order to understand if the generated proteins had biochemically relevant differences. Recombinant PPO corresponding to the native PPO accumulated at higher level and was more active than the two homologues. A cysteine present in the CTP seems to be able to modify the redox state of the enzyme and to be responsible for the alteration of the kinetic features. In contrast, the sensitivity to different herbicides was unaffected by modifications at the NH(2)-terminus, suggesting that the mode of action is non-competitive and that the NH(2)-terminus is involved in the recognition of the natural substrate.

Amino Acid Sequence↗