Log in with Facebook Log in with Google. Remember me on this computer. Enter the email address you signed up with and we'll email you a reset link. Need an account? Click here to sign up. Download Free PDF. Manuel Valdivia. A short summary of this paper. Received 1 September; revised 4 November ; accepted 10 November ABSTRACT Background: Infections due to Pseudomonas aeruginosa are among the leading causes of morbidity and mortality in patients who suffer from impaired immune responses and chronic diseases such as cystic fibrosis.
At present, aggressive antibiotic therapy is the only choice for management of P. Several P. However, none of them are currently available clinically. Methods: In this research, recombinant exoA-flagellin fliC fusion protein as a cocktail antigen was expressed and purified and its antigenic characteristics were evaluated. Results: Expression of recombinant fusion protein by E. Based on Western-blotting results, recombinant fusion protein showed a good antigenic interaction with sera from patients with various P.
Conclusion: These results suggested that recombinant exoA-fliC fusion protein can be produced in the laboratory, and tested as a candidate vaccine in P. This bacterium has different chromosomal and plasmid-mediated resistance genes seudomonas aeruginosa is a Gram-negative involved in the resistance of the bacterium to anti- P opportunistic pathogen that commonly infects patients with impaired immune response.
Even newly developed antibiotics have failed to reduce the mortality rate associated with this organism. Low permeability of outer membrane and different efflux pumps are also among receiving immunosuppressive drugs. The bacterium is common mechanisms of drug resistance in P. Hence, due to high prevalence of other clinical samples from nosocomial infections. Primer sequences were used in amplification of exotoxin A exoA and flagellin fliC gene fragments.
The toxin binds and Lithuania and pET22b Novagen, USA were used in enters into the cells by receptor-mediated endocytosis cloning and recombinant expression. Primers for exoA gene domains I-II, synthesis.
Also, exoA has ability to inhibit the host the binding and translocator domains were designed response to infection [9]. ExoA is an antigenic protein based on exoA gene sequence of P. Based on the results of different studies, these amino acids from fliC gene sequence of P. Therefore, exoA has aeruginosa M. Therefore, bp N-terminal been considered as a promising vaccine candidate for nucleotides of exoA gene and bp N-terminal pseudomonas infections []. Genomic DNA was extracted with The bacterial flagella are strong immunogenic factors phenol-chloroform method as described elsewhere and active or passive immunization with flagellar [17].
ExoA and fliC genes were amplified separately antigens induces antibody production. Overlapping PCR generating higher level of immune responses. Different was used for construction of exoA-fliC fusion gene cocktails or fusion proteins have been attempted [18]. Purification was performed under denaturing condition using 8 M urea for denaturation.
The contamination proteins were washed away from band related to fusion gene was cut and recovered by the column using wash buffer 20 mM sodium using a purification kit MN, Macherey Nagel, phosphate, mM NaCl, 50 mM imidazol, pH 8. The protein was then collected from column by using elution buffer 20 mM sodium phosphate, mM Cloning and sequencing.
Cloning of fusion gene NaCl and mM imidazol, pH 8. Western-blotting was used to sequenced Eurofins MWG Operon, Germany for evaluate the immunological properties of recombinant analysis of the sequence integrity. Then, the reactivity of recombinant protein E.
Then, the pET22b- patients with P. In an Hospital Tabriz. The P. HRP-conjugated anti-rabbit IgG for further four hours. For expressed protein solubility analysis, E. After 18 hours, it was gene from P. Recombinant exoA-fliC fusion protein was expressed as a kDa protein in high concentration. Western-blotting results. Western-blotting analysis showed that recombinant exoA-fliC was reacted with antibody against native exoA antibody Fig.
This finding verified the correct conformational structure of recombinant protein produced in E. Analysis for reactivity of recombinant fusion protein to sera from Fig.
Electrophoresis of overlapping PCR product on patients with P. Recombinant expression of exoA-flagellin fusion protein. For high-level expression of exoA-fliC fusion protein in E. Induction of E. Lane 1, standard protein size 1 2 3 marker; lane 2, pellet of un-induced bacteria; lane 3, pellet of IPTG-induced bacteria and lane 4, purified-recombinant fusion protein. Therefore, the management of P. Nowadays, development of alternative methods for treatment of P.
Various new Fig. Electrophoresis of digestion product. Lane 1 and 2, approaches have been investigated to combat pseudo- recombinant vector pET22b-exo-fliC digested whit BamhI and monas infection. For example, it has been shown that deletion of Glu in the toxic domain of exoA abolishes the toxicity of pseudomonas exoA [28]. In our study, non-toxic exoA with suitable immunogenic properties was obtained by deletion of enzymatic 70 kDa domain. In the recombinant method, not only production of large amounts of fliC is possible but also selection of conserved and immunogenic domains and deleting of unnecessary domains are feasible.
In our study, a bp fragment of fliC gene encoding conserved N-terminal amino acids of fliC and a bp fragment of exoA gene encoding N-terminal amino acids of exoA as non-toxic domain were selected and used for recombinant Fig.
Western-blot of recombinant fusion protein probed expression and protein production. Lane 1, In this research, exoA-fliC fusion gene was prepared pre-stained protein size marker and lane 2, purified fusion by overlapping PCR method and showed that this protein. This finding is consistent with the and preventive tools [4]. Several different antigens previous reports about advantages of overlapping PCR have been evaluated for their immunogenicity and for production of fusion genes [18, 30].
Therefore, it has been postulated that effective immune response induction may need the use of a cocktail of 1 2 3 4 5 6 different antigens [9, 21, 22]. Enhanced clearance of P. These studies proposed exoA and fliC of kDa P. The baseline-resolved, direct-MS spectra encouraged us to explore whether we could also characterize the antibodies produced directly from the crude media, by means of IM-MS.
The results indicate that despite the impurity of the sample, well-resolved IM-MS plots could be generated Supplementary Fig. The intact mass of the antibody and its corresponding CCS value could easily be extracted, emphasizing not only the additional experimental input yielded by this method, but also the fact that the direct-MS approach can be applied on various high-mass mass spectrometry platforms.
We next explored the G6 and G6 des13 pair of antibodies, examining whether the introduced mutations altered the overall structure of G6 des13 , in comparison to G6. IM-MS analysis demonstrated that, similar to the measurements conducted on the Orbitrap platform, the major charge series in the crude G6 sample corresponded to background medium proteins; nevertheless, the antibody signals could be clearly identified Fig.
In the G6 des13 sample, the most intense charge envelope corresponded to the antibody, while the medium protein signals were very low, supporting the view that G6 des13 exhibits a gain in expressibility.
Taken together, we found that direct-IM-MS measurements can therefore reflect the higher-order shape of unpurified antibodies. In addition, we examined whether it is possible to monitor changes in protein abundance within the crude media. To achieve this, a series of spectra were recorded using yeast, insect and human cultures supplemented with increasing concentrations of BSA.
Figure 4 shows representative mass spectra of the three different growth media, obtained using elevated BSA concentrations. The relative abundance of BSA was then estimated in respect to selected background peaks whose intensity remained unchanged throughout the analysis. For each mass spectrum, the intensity ratio of BSA and the selected reference peaks was calculated and plotted against known BSA concentrations Fig.
A linear trend line was observed for all three expression systems, however, the linearity range differed. We noticed that the range of linearity is dependent on the highest concentration level of BSA that can be quantified, which is limited by the detection of the reference peak. Taken together, the data indicates that for a specified range, it is possible to measure dynamic changes in protein levels directly from culture media. Changes in protein abundance can be monitored in crude media.
A series of measurements with different concentrations of externally-added BSA, were performed in growth media from yeast a , insect b , and human c cell cultures. The intensity ratio between BSA dark blue circles and background reference peaks labeled with asterisks was plotted against known BSA concentrations bottom graphs.
Trend lines and R-squared values are shown. Error bars represent standard deviations of three measurements. The original mean source data is shown in Supplementary Data 2. Here, we report a method for rapid characterization of secreted recombinant proteins, directly from crude growth media. Based on native-mass spectrometry, this method enables the assessment of multiple protein attributes in a single spectrum. We demonstrate that the expression, solubility and assembly state of overproduced proteins can be determined, as well as their associations with cofactors.
The mass accuracy afforded by this strategy enables not only the identification of post-translationally modified species, but also the existence of differential modifications, and the distribution of the different forms. We also show that the overall structure of the unpurified overproduced proteins can be defined.
Here, we have focused on recombinant proteins produced in yeast, insect, and human cells; however the method can be expanded to include many other expression platforms that overproduce secreted proteins, such as Chinese hamster ovary cells CHO 33 and murine myeloma cell lines GS-NS0 10 , The fundamental requirement for this approach, however, is that the overexpressed target becomes the dominant protein in the growth medium.
Consequently, the in-depth structural characterization afforded by native mass spectrometry analysis can be performed. Moreover, it should be noted that the strategy is not dependent on a specific mass spectrometer platform; rather, it is a general approach that can be applied on either Orbitrap or QTOF-based platforms that provide extended mass range 35 , 36 , A key to successful production of recombinant proteins for both industrial and pharmaceutical applications is to achieve maximum productivity at a reasonable cost 10 , Therefore, a screening program is generally performed to identify high-producing cells, optimize genetic and engineered constructs, and improve media formulation.
We anticipate that the method outlined here will facilitate this labor-intensive and costly process, by overcoming the need for protein purification prior to characterization, and by providing, in a single step, in-depth quality assessment of the protein produced. In addition, the minimal amount of sample required, coupled with the simplicity and feasibility of the approach across different platforms, also make it suitable for high-throughput screening procedures that may further reduce the time gap between production and characterization.
The ability to monitor, in real time, recombinant protein production is a valuable feature of this strategy. Therefore, we expect that further extension of this method will involve its coupling to microfluidics systems that will enable continued analysis of cultures grown in bioreactors. Such analyses will make it possible to capture optimal harvesting times, preventing misfolding and proteolysis, and determine the necessity of cofactor supplements to ensure proper folding and assembly.
Such knowledge will improve both yield and quality of the recombinant proteins produced. Yeast: CBM3a, the cellulose-specific carbohydrate-binding module of the scaffoldin protein from C. Genomic integration into P. Insect cells: A modified monomeric ectodomain of the N. Sf9 cells were co-transfected with 0. Four days later medium was collected from transfected cells P 0 , and was used to infect fresh Sf9 cells for amplification P 1 —P 2. Human cells: The variable regions of the heavy and light chains of several synthetic chimeric antibodies were cloned separately, upstream of the IgG1 human Ab scaffold, into p3BNC plasmids containing the human IgG secretion signal peptide.
The difference between G6 and its designed variant G6 des13 are six amino acids in the interface between the light and heavy chains; b Antibodies D The difference between D Yeast: CBM3a was expressed in P. Serum albumin harboring its native secretion signal was expressed in P. Expression was induced by the addition of 0.
Insect cells: TfR1 was expressed in baculovirus-infected Sf9 cells. Human cells: For antibody expression and secretion, two plasmids for light and heavy chains were co-transfected together simultaneously into cells.
Antibodies were expressed in either adherent or suspension-grown HEK cells. Growth media were collected after 5—7 days. We found experimentally that performing 1—2 cycles of buffer exchange into high salt ammonium acetate efficiently replaces the bulk non-volatile compounds in the sample, and result in high quality spectra. CBM3a and serum albumin: P. In order to reduce cysteine bonds, we tested different concentrations of the reducing agents DTT and TCEP, added directly to the growth medium, at different incubation times.
All measurements were performed at least three times. Spectra recorded by the Orbitrap platform were converted to Masslynx-compatible files using the Waters DataBridge software. All spectra were analyzed using the MassLynx V4. Reported masses shown in Daltons and mass errors were calculated using MassLynx. Top-down proteomic analysis was performed using the ProSightPC v. The following experimental parameters were used on the Orbitrap platform: All spectra are shown without smoothing, and the instrument was calibrated externally, using cesium iodide.
Conditions within the mass spectrometer were adjusted to preserve noncovalent interactions, and adjusted according to the type of protein measured. The source was operated in positive mode, capillary voltage was set to 1. MS spectra were recorded at a resolution of 10,—70,, according to the complexity of the sample.
Raw spectra were deconvoluted using Deconvolution 4 protein software Thermo. Bent flatapole DC bias and axial gradient were set to 1. For the measurement of intact glycans, no HCD voltage was applied. Trapping gas was set to a pressure of 1, which corresponds to HV pressure of 9. The following experimental parameters were used on the Synapt G2 platform for IM-MS measurements: All spectra are shown with minimal smoothing, and the instrument was calibrated externally, using cesium iodide.
For CBM3a measurements, instrument parameters were set as follows: Capillary voltage of 1. No trap and transfer collision energies were applied. For antibody measurements, instrument parameters were set as follows: Capillary voltage of 1. No transfer collision energy was applied. Proteins used for calibrations were: Ubiquitin 8. All measurements were conducted on the same day; the only parameters modified for the test and calibrating proteins were capillary voltages.
Spectra were recorded on an Orbitrap instrument, as described for serum albumin. The relative values obtained were plotted against known BSA concentrations. Each experiment was performed at least three times. The datasets generated during the current study are available from the corresponding author on reasonable request.
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