Small-scale Purification Research Articles

Small scale purification of constituents from complex natural product extracts using sub-2-µm chromatography

Extracts from natural product samples can be complex often containing a large number of diverse compounds. Increased separation performance of sub-2-μm column technology along with low dispersion instrumentation provides a tool that produces sharp, narrow, and more concentrated peaks. When there is a need to collect narrow peaks from these complex mixtures, traditional fraction collection instrumentation designed for preparative HPLC conditions does not provide an adequate solution primarily due to peak broadening through collection valves and tubing.

Tail proteins of phage T5: Investigation of the effect of the His6-tag position, from expression to crystallisation

Upon binding to its bacterial host receptor, the tail tip of phage T5 perforates, by an unknown mechanism, the heavily armoured cell wall of the host. This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions. In the perspective of a structural study of the phage tail, we have systematically overproduced eight of the eleven T5 tail proteins, with or without a N- or a C-terminal His6-tag. The widely used Hi6-tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography. The presence of a tag however is not always innocuous. We combined automated gene cloning and expression tests to rapidly identify the most promising constructs for proteins of phage T5 tail, and performed biochemical and biophysical characterisation and crystallisation screening on available proteins. Automated small-scale purification was adapted for two highly expressed proteins. We obtained structural information for three of the proteins. We showed that the presence of a His6-tag can have drastic effect on protein expression, solubility, oligomerisation propensity and crystal quality.

In vivo antigen-driven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells.

The ability to rapidly generate large panels of antigen-specific human antibodies in a rodent would enable the efficient discovery of novel therapeutically useful antibodies. We have developed a system wherein human antigen-specific antibody-secreting plasmablasts can be enriched in vivo, in a severe combined immunodeficient (SCID)/beige mouse host. The antigen-specific plasmablasts can then be sorted by flow cytometry, enabling single-cell cloning and expression of fully human immunoglobulin-G. By using this technique, we have generated four broadly reactive anti-influenza A antibodies. Therefore, the method described here is useful for the identification of rare functional antibodies. This protocol takes ∼1 month to complete, from the time of human vaccination to the cloning of heavy- and light-chain genes. For additional small-scale transient expression, purification and binding analysis, the protocol would take an additional month.

High‐throughput ion exchange purification of positively charged recombinant protein in the presence of negatively charged dextran sulfate

Product quality analyses are critical for developing cell line and bioprocess producing therapeutic proteins with desired critical product quality attributes. To facilitate these analyses, a high-throughput small-scale protein purification (SSP) is required to quickly purify many samples in parallel. Here we develop an SSP using ion exchange resins to purify a positively charged recombinant growth factor P1 in the presence of negatively charged dextran sulfate supplemented to improve the cell culture performance. The major challenge in this work is that the strong ionic interaction between P1 and dextran sulfate disrupts interaction between P1 and chromatography resins. To solve this problem, we develop a two-step SSP using Q Sepharose Fast Flow (QFF) and SP Sepharose XL (SPXL) resins to purify P1. The overall yield of this two-step SSP is 78%. Moreover, the SSP does not affect the critical product quality attributes. The SSP was critical for developing the cell line and process producing P1.

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness1. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST)2,3 and a C-terminal 10xHis tag4, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression5. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme6. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

GST-His purification: A Two-step Affinity Purification Protocol Yielding Full-length Purified Proteins

Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.

Automated disposable small scale reactor for high throughput bioprocess development: A proof of concept study

The acceleration of bioprocess development for biologics and vaccines can be enabled by automated high throughput technologies. This will alleviate the significant resource burden from the multi-factorial statistical experimentation required for controlling product quality attributes of complex biologics. Recent technology advances have improved clone evaluation and screening, but have struggled to combine the scale down criteria required for both high cell density cell culture and microbial processes, with sufficient automation and disposable technologies to accelerate process development. This article describes the proof of concept evaluations of an automated disposable small scale reactor for high throughput upstream process development. Characterization studies established the small scale stirred tank disposable 250 mL reactor as similar to those of lab and pilot scale. The reactor generated equivalent process performance for industrial biologics processes for therapeutic protein and monoclonal antibody production using CHO cell culture, Pichia pastoris and E. coli. This included similar growth, cell viability, product titer, and product quality. The technology was shown to be robust across multiple runs and met the requirements for the ability to run high cell density processes (>400 g/L wet cell weight) with exponential feeds and sophisticated event triggered processes. Combining this reactor into an automated array of reactors will ultimately be part of a high throughput process development strategy. This will combine upstream, small scale purification with rapid analytics that will dramatically shorten timelines and costs of developing biological processes.

Pesticides Removal by Filtration over Cactus Pear Leaves: A Cheap and Natural Method for Small-Scale Water Purification in Semi-Arid Regions

This study aims to examine the efficiency of Opuntia ficus-indica for removing organochlorine pesticides from surface waters. Adsorption properties such as size, dose, and time of O. ficus-indica for aldrin, dieldrin, and dichlorodiphenyltrichloroethane (DDT)were studied through stirring and column methods. Because of their high affinity and swelling characteristics, dried O. ficus-indica was studied in stirring while fresh unpeeled O. ficus-indica was applied in both stirring and column experiments and proved to be well-suited to column application. Before removing pesticides, the column was flashed with distilled water eliminate the turbidity and smell from fresh unpeeled cactus. The removal of pesticides increased with an increasing adsorbent dose and decreased with adsorbent particle sizes. The optimum adsorbent dose is 10 g for dried and 15 g for fresh unpeeled O. ficus-indica. The experimental results show that O. ficus-indica possesses strong adsorption ability for aldrin, dieldrin, and DDT, and the adsorption isotherm data obeyed the Freundlich model. The results of our small-scale experiments suggest a strong potential to develop local small-scale water treatment units that can be used at the level of individual households or local communities, using a widely available adsorbent.

Small-scale purification and mass spectrometry analysis reveal a third aquaporin-4 protein isoform of 36 kDa in rat brain

Aquaporin-4 (AQP4) is known to have two main isoforms M1 and M23 in the brain. Immunoblot analyses have provided evidence of additional AQP4 immunopositive bands, suggesting that the repertoire of AQP4 isoforms is broader than previously assumed. As isoforms beyond M1 and M23 are not observed in recombinant systems, investigation of novel isoforms requires the use of a native source. Here we report purification of AQP4 to three silver-stained proteins on SDS-PAGE. This was achieved by organelle separation, alkaline stripping of cellular membranes, detergent solubilization and multiple chromatographic steps. The three proteins that co-purified were identified as AQP4 by mass spectrometry. These results represent the first purification of AQP4 from a native source and demonstrate by mass spectrometry the presence of a third AQP4 isoform of 36kDa in the rat brain. Immunoblots revealed that the same isoform is present in the mouse, pig, and human brain.

Purification of Adenovirus by Cesium Chloride Density Gradients

Adenovirus are efficient gene delivery systems. The standard method for purification of adenoviral vectors is based on using a cesium chloride (CsCl) density gradient combined with ultracentrifugation. This method is suitable for small-scale purification and is less expensive than column chromatography or commercial purification kits., 腺病毒是有效的基因递送系统。 用于纯化腺病毒载体的标准方法基于使用氯化铯（CsCl）密度梯度与超速离心的组合。 该方法适合于小规模纯化，并且比柱层析或商业纯化试剂盒更便宜。

Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures

The quality and yield of extracted DNA are critical for the majority of downstream applications in molecular biology. Moreover, molecular techniques such as quantitative real-time PCR (qPCR) are becoming increasingly widespread; thus, validation and cross-laboratory comparison of data require standardization of upstream experimental procedures. DNA extraction methods depend on the type and size of starting material(s) used. As such, the extraction of template DNA is arguably the most significant variable when cross-comparing data from different laboratories. Here, we describe a reliable, inexpensive and rapid method of DNA purification that is equally applicable to small or large scale or high-throughput purification of DNA. The protocol relies on a CTAB-based buffer for cell lysis and further purification of DNA with phenol : chloroform : isoamyl alcohol. The protocol has been used successfully for DNA purification from rumen fluid and plant cells. Moreover, after slight alterations, the same protocol was used for large-scale extraction of DNA from pure cultures of Gram-positive and Gram-negative bacteria. The yield of the DNA obtained with this method exceeded that from the same samples using commercial kits, and the quality was confirmed by successful qPCR applications.

Establishment and application of a tandem affinity purification system of innate immune regulatory protein PKR

Objective To establish a tandem affinity purification(TAP) system of innate immune-regulatory protein PKR and analyze PKR function, for the future screen and identification of novel PKR-interaction proteins. Methods PKR gene was amplified by PCR, and then cloned into a mammalian expression vector pcTAP-A. Recombinant pcTAP-PKR was transfected into PKR knock-down(PKRkd) HeLa cells by LipofectAMINE 2000,and the PKR overexpressed HeLa cells were harvested for mitogen-activated protein kinases(MAPK) activation analysis. Cell extracts of PKR overexpressed cells were purified using TAP kit and examined by Western blot. Results Cal modulin resin(CBP) and streptavidin resin(SBP) tagged PKR was detected in PKRkd HeLa cells as early as 24 h upon transfection with pcTAP-PKR, and its expression decreased at later time points. The overexpression of PKR was autophosphorylated, and thus involved in the regulation of MAPK actviation. After small-scale TAP kit purification, PKR protein was detectable by Western blot. Conclusion We have successfully established a TAP system that over-expresses functional PKR, providing a useful tool for the future study on the identification of PKR interacting proteins. Key words: Double-stranded RNA; Protein kinase PKR; Affinity; Purification

In this issue

PROTEOMICSVolume 11, Issue 13 In this issue In this issue First published: 27 June 2011 https://doi.org/10.1002/pmic.201190060AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Mix or match? BOGO (Buy one, get one (free, except in countries where restricted)) The idea of “one free” appeals to most consumers (and scientists, too) but normally only if they need two of the items. In this case it is multiple protein or peptide identifications and small-scale purification of selected molecules from complex mixtures. Dunham et al. were in need of more efficient methods to process multiple samples, preferably affinity purified. They report here on comparison of four proteins with different binding partners and complexes: COPS5, eIF4A2, RAF1, and MEPCE. COPS5 has been identified as part of a ubiquitin (COP9) signalosome; RAF1 is a serine-threonine protein kinase, part of the MAP kinase ERK pathway; MEPCE is the 7SK snRNA. Also involved are chaperones, translation initiation factors and RNA processing factors. Each was fractionated classically and multidimensionally (FLAG) and results analyzed for efficiency. And the winner is… pp. 2603–2612 Starting small: Phorbol esters as a model breast cancer system So many parameters, so little time. Particularly cell–cell-based systems. How about those small molecules, like phorbol esters, able to turn cancers on by turning steroid receptors off? (Add the ability to shut off HER-2 and you have a triple negative breast cancer, one of the toughest breast tumor types.) Dolai et al. report here on controls of a related tumor recently in the news. The bisindolylmaleimide (Bis)-binding molecules have been identified as protein kinase C inhibitors in the MDA-MB-231 model breast cancer system. Gene ontology analysis of the 174 Bis-stimulated proteins captured revealed 42% ATP and 6% GTP binding and 21% nucleoside-triphosphatase activity. Novel evidence added glyceraldehyde-3-phosphate dehydrogenase, nucleolar RNA helicase-2, and heterogeneous nuclear ribonucleoprotein M to the list of regulated enzymes. pp. 2683–2692 One-step dance speeds through the cousins When social obligations require that you dance with all of your younger cousins (and your mother does mean all), colluding with the band leader/DJ to “keep 'em short” will save you time and energy and get you to the poker game sooner. Araki et al. have done just that for developing a clinical test for pregnancy-induced hypertension (PIH). A number of pathological abnormalities have been assigned to PIH, including dysfunction of the placental endothelium and exaggerated systemic inflammatory response. Its cause is clearly multifactorial but, currently, the only diagnostic is monitoring blood pressure. New technology to the rescue! The “peptidome” is defined by the proteomic pattern of peptides and this group has reported major improvements to MALDI/TOF precision and a single step 1-D electro-blotting procedure. During the loading of the “BlotChip,” overabundant species, except cousins, can be selectively avoided. pp. 2727–2737 Volume11, Issue13No. 13 July 2011 RelatedInformation

Rapid characterization of N‐linked glycans from secreted and gel‐purified monoclonal antibodies using MALDI‐ToF mass spectrometry

Recombinant monoclonal antibodies (MAbs) are increasingly being used for therapeutic use and correct glycosylation of these MAbs is essential for their correct function. Glycosylation profiles are host cell- and antibody class-dependent and can change over culture time and environmental conditions. Therefore, rapid monitoring of glycan addition/status is of great importance for process validity. We describe two workflows of generally applicability for glycan profiling of purified and gel-purified MAbs produced in NS0 and CHO cells, in which small-scale antibody purification and buffer exchange is combined with PNGase F glycan cleavage and graphite HyperCarb desalting. MALDI-ToF mass spectrometry is used for sensitive detection of glycan forms, with the ability to confirm glycan structures by selective ion fragmentation. Both workflows are rapid, technically simple and amenable to automation, and use in multi-well formats.

MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms

BackgroundThe production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms.ResultsWe present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented.ConclusionsThe objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation.

Electrospun polyethersulfone affinity membrane: Membrane preparation and performance evaluation

Non-woven polyethersulfone (PES) membranes were prepared by electrospinning. After heat treatment and surface activation, the membranes were covalently functionalized with ligands to be used as affinity membranes. The membranes were characterized in terms of fiber diameter, porosity, specific area, pore size, ligand density and binding capacities. To evaluate the binding efficiency of the membrane, dynamic adsorption of bovine serum albumin (BSA) on the Cibacron blue F3GA (CB) functionalized PES membrane was studied. Experimental breakthrough curves were fitted with the theoretical curves based on the plate model to estimate plate height ( H p ) of the affinity membrane. The high value of H p (1.6–8 cm) of the affinity membrane implied a poor dynamic binding efficiency, which can be explained by the intrinsic microstructures of the material. Although the electrospun membrane might not be an ideal candidate for the preparative affinity membrane chromatography for large-scale production, it still can be used for fast small-scale protein purification in which a highly efficient binding is not required. Spin columns packed with protein A/G immobilized PES membranes were demonstrated to be capable of binding IgG specifically. SDS-PAGE results demonstrated that the PES affinity membrane had high specific binding selectivity for IgG molecules and low non-specific protein adsorption. Compared with other reported affinity membranes, the PES affinity membrane had a comparable IgG binding capacity of 4.5 mg/ml, and had a lower flow through pressure drop due to its larger pore size. In conclusion, the novel PES affinity membrane is an ideal spin column packing material for fast protein purification.

One-step chromatographic purification of human papillomavirus type 16 L1 protein from Saccharomyces cerevisiae

The prophylactic human papillomavirus vaccine is based on recombinant L1 protein produced in yeast or insect cells. L1 is a major capsid protein that self-assembles into virus-like particles (VLP). Conventionally, several chromatography steps are required to purify it; the steps are time consuming, and they result in losses of the target protein. Ultracentrifugation using a sucrose cushions or cesium chloride density gradients, and size-exclusion chromatography, has also been routinely used for small scale purification of L1 protein. However, these methods require a great deal of time and labor, and are not suitable for industrial-scale purification. To resolve these problems, we have developed two simple one-step chromatography methods for purifying recombinant HPV16 L1 protein produced in Saccharomyces cerevisiae. Eighty percent of the contaminating protein was removed by ammonium sulfate precipitation and by precipitating contaminants prior to the chromatography step. One method uses heparin chromatography and the other, cation-exchange chromatography, and recoveries by the two methods were both about 60%, the highest recoveries of L1 protein achieved so far. We confirmed that HPV16 L1 protein purified by either method self-assembles into VLP. We anticipate that these one-step chromatography methods will reduce the time, cost and labor needed for purification of L1 protein, and facilitate the study of prophylactic HPV vaccines.

Docking of Antizyme to Ornithine Decarboxylase and Antizyme Inhibitor using Experimental Mutant and Double-Mutant Cycle Data

Antizyme (Az) is a highly conserved key regulatory protein bearing a major role in regulating polyamine levels in the cell. It has the ability to bind and inhibit ornithine decarboxylase (ODC), targeting it for degradation. Az inhibitor (AzI) impairs the activity of Az. In this study, we mapped the binding sites of ODC and AzI on Az using Ala scan mutagenesis and generated models of the two complexes by constrained computational docking. In order to scan a large number of mutants in a short time, we developed a workflow combining high-throughput mutagenesis, small-scale parallel partial purification of His-tagged proteins and their immobilization on a tris-nitrilotriacetic-acid-coated surface plasmon resonance chip. This combination of techniques resulted in a significant reduction in time for production and measurement of large numbers of mutant proteins. The data-driven docking results suggest that both proteins occupy the same binding site on Az, with Az binding within a large groove in AzI and ODC. However, single-mutant data provide information concerning the location of the binding sites only, not on their relative orientations. Therefore, we generated a large number of double-mutant cycles between residues on Az and ODC and used the resulting interaction energies to restrict docking. The model of the complex is well defined and accounts for the mutant data generated here, and previously determined biochemical data for this system. Insights on the structure and function of the complexes, as well as general aspects of the method, are discussed.

A three-phase microfluidic chip for rapid sample clean-up of alkaloids from plant extracts

A three-phase microchip was developed for the rapid and efficient small-scale purification of alkaloids from plant extracts. As part of the development of such a three-phase microchip, first a two-phase microchip with two channels (3.2 cm and 9.3 cm) was used to study the extraction efficiency of strychnine nitrate and strychnine at various flow rates. Strychnine was extracted from a basic aqueous phase to a chloroform phase (extraction) or strychnine was extracted from a chloroform phase into an acidic aqueous phase (back extraction). Subsequently, the "simultaneous extraction and back extraction" of strychnine was carried out in a three-phase microchip. The experimental extraction rate and yield were compared with model data. At a residence time of 25 sec, 79.5% of strychnine was extracted into the acidic aqueous phase using the three-phase microchip. In general, a good correlation was found between experimental results and model data for both two- and three-phase extractions. Finally, the three-phase microchip was employed in the purification of alkaloids (strychnine and brucine) from Strychnos seed extracts.

Identification of Highly Expressed, Soluble Proteins using an Improved, High-Throughput Pooled ORF Expression Technology

This article describes an improved pooled open reading frame (ORF) expression technology (POET) that uses recombinational cloning and solution-based tandem mass spectrometry (MS/MS) to identify ORFs that yield high levels of soluble, purified protein when expressed in Escherichia coli. Using this method, three identical pools of 512 human ORFs were subcloned, purified, and transfected into three separate E. coli cultures. After bulk expression and purification, the proteins from the three separate pools were digested into tryptic peptides. Each of these samples was subsequently analyzed in triplicate using reversed-phase high-performance liquid chromatography (LC) coupled directly online with MS/MS. The abundance of each protein was determined by calculating the average exponentially modified protein abundance index (emPAI) of each protein across the three protein pools. Human proteins that consistently gave high emPAI values were subjected to small-scale expression and purification. These clones showed high levels of expression of soluble protein. Conversely, proteins that were not observed by LC-MS/MS did not show any detectable soluble expression in small-scale validation studies. Using this improved POET method allows the expression characteristics of hundreds of proteins to be quickly determined in a single experiment.