Leonard K Pattenden 2 ,. Access enabled via: An Institution. PDF Full text Related articles. Abstract Maltose-binding protein MBP is a carrier protein for high level recombinant protein and peptide production from either the cytoplasm or periplasm of Escherichia coli. The affinity matrix for purifying MBP-passenger proteins utilizes amylose … more. Citations 5. Recent citations: Claudia Ortega et al.
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Howells , , Springer Protocols. Reconstitution of S. Lima , , Springer Protocols. See more. Glover , , Springer Protocols. References Maina, C. Addgene, ,. Nallamsetty, S. Luecke, h. Pflugrath, J. Bruns, C. Kang, C. Oh, B. Sack, J. Yao, N. Nickitenko, A.
Sleigh, S. Quiocho, F. Boos, W. Kapust, R. Richarme, G. The approach should also be easily adaptable to robotization. It would be interesting to automatize this protocol for purifying at high throughput families of proteins, which, like E6 oncoproteins, are difficult to fold except when fused to MBP or other soluble carrier proteins. The overexpression of such constructs leads to a limited proportion of properly folded and active monomers that require to be separated from non-active oligomers, which is now possible using the protocol presented here.
Expression was induced by adding 0. Purification buffers were thoroughly degassed and equilibrated with argon before adding 2 mM DTT. This buffer was supplemented with 10 mM imidazole for purification on Nickel resins. Pellet equivalent to mL culture volume was resuspended in 10 mL lysis buffer. The incubation time of the cleared lysate with the resin was 2 h in the initial purification tests, before to be evaluated as a variable parameter 5 min, 30 min or 2 h and reduced to 5 min in the final optimized protocol.
The supernatant was recovered and used as final purified protein solution. Protein concentration was determined by Nanodrop measurement, based on absorbance at nm. Final protein samples were systematically analyzed either by SDS-PAGE or by microfluidics capillary gel electrophoresis in order to check the presence of protein contaminants in denaturing conditions. Purification samples from the washing steps and elution were transferred in well plates, mixed with the Caliper sample buffer and boiled according to the manufacturer instructions.
The kit allows to label the proteins with a fluorescent dye. This dye is excited by a laser during protein capillary electrophoresis in denaturing conditions.
The emitted fluorescence signal is plotted versus migration time, leading to the protein separation according to their molecular weight. Migration time is first converted into molecular weight using standard protein markers before a quantitative analysis is performed thanks to empirical linear correlation between fluorescence intensity and protein amount. Spikes of absorbance nm signal due to air bubbles were excluded from the chromatogram before performing data analysis. The original chromatograms are provided in Additional file 2.
Mix 2 contained ferritine 0. The resulting elution volumes were used to establish a calibration plot to determine the molecular weight MW , based on the following equation [ 33 ]:. Thus, the coefficients a and b were determined by linear regression using the following equation [ 33 ]:. The area of elution peaks based on absorbance at nm was calculated using the evaluation module of Unicorn 5. According to the column calibration, we inferred the elution volume of monomeric approximately 1.
For each assay, the area of the elution peak corresponding to the monomer was divided by the area of peaks corresponding to all eluted protein species monomeric and oligomeric , allowing us to measure the ratio of monomer and oligomer in the purified protein sample.
This uncertainty, although given as a percentage, corresponds to the absolute variation of the ratio of monomeric protein and not as a relative variation coefficient. Briefly, the chip is coated with a deoxyribooligonucleotide that hybridizes with the complementary strand bound to streptavidin. The biotinylated peptide binds to streptavidin and can be washed by dehybridizing the two oligonucleotides.
An empty control surface was systematically included on every cycle to serve as a reference for non-specific binding of the analyte to the matrix and for monitoring changes in solution refractive index.
This reference surface was treated as the peptide surfaces except that peptide injection was omitted. The SPR signals from the regions corresponding to the protein injection and post-injection phases were plotted as RU versus time. Data were first processed using the Biacore T Evaluation 1.
Sensorgrams obtained for the different protein concentrations were corrected for buffer effects and bulk refractive index changes by subtracting the empty cell signal, and subsequently normalized according to the peptide levels which can slightly differ from cycle to cycle due to the immobilization process. The steady-state binding signal was derived by averaging the signals at equilibrium within a five second-window R eq.
Steady-state analysis was performed using an in-house Python script by fitting the average and normalized signal R eq as a function of total analyte concentration, and assuming a simple interaction binding isotherm model. Uncertainties of the derived parameters were estimated using Monte-Carlo approach, considering an experimental uncertainty of 5 RU. This value was estimated by duplicating the full experimental cycle corresponding to a single protein concentration.
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Attachment of group-containing ligands to insoluble polymers by means of bifunctional oxiranes. Download references. All authors read and approved the final manuscript. The authors thank D. Altschuh for the optimization of Biacore assays, P. Eberling for peptide synthesis, A. Vanden Broeck for his valuable help on size-exclusion chromatography columns, P. C and P. Other authors declare that they have no competing interests. The datasets analyzed during the current study are included within the article and additional files.
The authors declare that the content is solely their responsibility and does not represent the official views of the National Institutes of Health. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. You can also search for this author in PubMed Google Scholar. Corrected size-exclusion chromatograms. Prior to protein peak integration, spikes due to air bubbles were excluded. In order to control to oligomeric state of the purified protein, we performed systematic analytical SEC on the final protein sample.
The calibration of the column allowed us to estimate the size of the particles at different elution volumes, thus the elution peak of monomeric protein is indicated by an arrow. On this protein challenging to purify, the increase of the monomer fraction indicated by an arrow between the two elution methods is dramatic.
The elution peak of monomeric protein is indicated by an arrow. The decrease of the oligomeric fraction eluted between the void volume V 0 and the monomer fraction is particularly visible for nickel resin N1. Raw size-exclusion chromatograms. We provide in this file the raw data of size-exclusion chromatograms used in the study absorbance at nm versus elution volume. The first worksheet entitled Readme contains explanations on the nomenclature used for worksheet titles.
Reprints and Permissions. Bonhoure, A. One-step affinity purification of fusion proteins with optimal monodispersity and biological activity: application to aggregation-prone HPV E6 proteins.
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Skip to main content. Search all BMC articles Search. Download PDF. Abstract Background Bacterial expression and purification of recombinant proteins under homogeneous active form is often challenging. Results In this study, we took advantage of the distinct size and diffusion properties of MBP-E6 monomers and oligomers to separate these two species using a rapid batch preparation protocol on affinity resins. Conclusions We have designed a rapid single-step batch affinity purification approach to isolate biologically active monomers of MBP-fused E6 proteins.
Background Protein solubility is a major issue in recombinant protein purification from Escherichia coli. Results Protein constructs used for this study To develop and evaluate a new E6 purification protocol, we used two distinct HPV E6 oncoproteins.
Full size image. Table 1 Panel of resins used for affinity purification, gathering technical data provided by the manufacturers Full size table. Discussion Quality of recombinant protein sample can be improved by optimizing either the expression or the purification process. Conclusions In the present work, we designed a single-step purification strategy optimized for maximal recovery of monomeric MBP-E6 that we accurately quantified by means of analytical SEC.
Microfluidics capillary gel electrophoresis Purification samples from the washing steps and elution were transferred in well plates, mixed with the Caliper sample buffer and boiled according to the manufacturer instructions. References 1. PubMed Google Scholar 2. Google Scholar 5.
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